JP2002218977A - Method for recombinant gene formation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for recombinant gene formation, with which a recombinant gene can be formed in the body of a microorganism, the function of the recombinant gene is evaluated in the body of microorganism as it is and an objective gene can be directly searched. SOLUTION: This method for recombinant gene formation is a method for recombining two or more kinds of genes having homology in the same body of microorganism in which a first plasmid vector obtained by connecting one gene to a specific base sequence derived from Bacillus sp.KSM-KP43, a base sequence in which one or more bases are deleted or substituted or added in the base sequence or a base sequence encoding a specified amino acid sequence different from that of the base sequence derived from Bacillus sp.KSM-KP43 or an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence and the other plasmid vector obtained by connecting the other gene to a replication initiation domain different from the first plasmid vector are transferred to a microorganism and homologous recombination is carried out, between the genes.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for forming a recombinant gene in a microorganism.

[0002]

2. Description of the Related Art In recent years,
Research on gene analysis, including elucidation of genomic information of various organisms, has been actively conducted, and accordingly, genetic engineering and protein engineering have become increasingly important. Protein,
The three-dimensional structures of DNA, RNA and their complexes have been estimated, and the field of elucidating the relationship between the structure and function of functional molecules such as enzymes has been advanced. In developing these functional molecules, how diverse the target functional molecules should be,
A very important point is how to efficiently search for molecules that meet the purpose from these diverse molecules.

[0003] As a method for imparting diversity to a gene, a method of randomly introducing a mutation into a target gene sequence has been established. As methods for introducing mutations at random, a method using a chemical modifier and a method using gene amplification by polymerase chain reaction are considered to be the main methods.

However, in the random mutation method, in order to cause recombination in a test tube to give diversity to a target gene, these recombinant genes are returned one by one to the living body (in a microorganism) and adapted to the purpose. An extra operation of checking whether or not it is necessary is required. In particular, there is a problem that it is extremely inefficient to perform this operation on a host cell having a low transformation rate by a foreign gene or a host cell having a poor growth.

[0005] The present invention provides a system for evaluating the function of a recombinant gene in a microorganism and directly searching for the gene of interest in the microorganism when the gene of interest is given diversity by genetic recombination. An object of the present invention is to provide a method for forming a transgene.

[0006]

Means for Solving the Problems The present inventors have studied the production of a recombinant gene, and as a result, have found that a plasmid vector containing a DNA fragment having a specific replication initiation region and a heterologous vector having a different replication initiation region. By including homologous genes in each of the plasmid vectors and coexisting in microorganisms, gene recombination occurs by the homology region existing between the genes, and it is possible to form diverse recombinant genes. I found it.

That is, the present invention relates to a method for recombining two or more homologous genes in the same microorganism, wherein one gene has the nucleotide sequence shown in SEQ ID NO: 1 or one or more nucleotides in the nucleotide sequence. A base sequence in which a base is deleted, substituted or added, or an amino acid sequence represented by SEQ ID NO: 2 or a base sequence encoding an amino acid sequence in which one or more amino acids are deleted, substituted or added to the amino acid sequence Recombinant gene that introduces a first plasmid vector and another plasmid vector in which another gene is linked to a replication initiation region different from that of the first plasmid vector into a microorganism and causes homologous recombination between the genes The present invention provides a forming method.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The first plasmid vector of the present invention comprises a base sequence of one gene represented by SEQ ID NO: 1 or a base sequence in which one or more bases are deleted, substituted or added to the base sequence. It refers to a DNA fragment or a plasmid vector linked to a base sequence encoding an amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence in which one or more amino acids have been deleted, substituted or added to the amino acid sequence.

[0009] "One gene" is a gene to be imparted with diversity by genetic recombination according to the method of the present invention, such as a gene encoding various enzymes, various functions in vivo, and regulation of metabolism. Bioactive peptides such as amylase, protease, cellulase, lipase, pectinase, pullulanase, peroxidase, oxygenase, catalase, etc., insulin, human growth hormone, interferon, calcitonin, interleukin, etc. And genes encoding proteins involved in antibiotic resistance.

[0010] These genes are each expressed in the living body or show some effect on the biological function. Therefore, if diversity is provided in the microorganism, the effect can be detected immediately, and selection of the target gene is very difficult. It is done efficiently.

[0011] Further, the first plasmid vector may have a nucleotide sequence represented by SEQ ID NO: 1 or a nucleotide sequence in which one or more bases have been deleted, substituted or added thereto, or an amino acid sequence represented by SEQ ID NO: 2 or It has a base sequence encoding an amino acid sequence in which one or more amino acids have been deleted, substituted or added to the amino acid sequence. Such a nucleotide sequence indicates a replication initiation region, which is a so-called RCR.
(Rolling circle-replicating) form (Gros, MF et
al., The EMBO Journal, 6, 3863-3869, (1987)). Further, the first plasmid vector having the nucleotide sequence is a plasmid pIP404 (Garnier, T. and Cole, ST, Plasmid,
19,134-150 (1988)), a novel plasmid having about 40% homology with the amino acid sequence of the replication initiation region and less than 20% homology with the replication initiation region of the other plasmids. .

[0012] The degree of mutation of the region in the first plasmid is determined by amino acids 1 to 432 in SEQ ID NO: 2.
Preferably have a homology of 40% or more,
More preferably, it has a homology of 60% or more,
It is particularly preferred that they have a homology of 70% or more.
In addition, "one or more" means one or more or more. Preferred examples of the plasmid vector having the base sequence include, for example, Bacillus sp. KSM-KP43 strain (FERM BP-6532).
Derived plasmid DNA.

The plasmid DNA for constructing the first plasmid vector of the present invention can be obtained, for example, by an alkali extraction method (Birnboim and Doly 1979 Nucleic Aci) from Bacillus sp. Strain KSM-KP43 (FERM BP-6532).
ds Res., 7: 1513-1523) and the PCR method of synthesizing appropriate primers can be used to isolate and obtain, and to initiate replication of the obtained plasmid DNA. The first plasmid vector of the present invention can be constructed by ligating the DNA fragment containing the region and the gene for the above modification.

In the present invention, at least one other plasmid vector different from the first plasmid vector is used in addition to the first plasmid vector. Such “another plasmid vector” has a replication initiation region different from that of the first plasmid vector and can coexist with the plasmid vector in a microorganism, and is identical to one gene present on the first plasmid vector. A gene having homology is linked to the replication initiation region. The replication initiation region of the other plasmid vector is not particularly limited, but is preferably of the type that forms an RCR form. By using a plasmid vector having such a replication initiation region, Can coexist more stably.

As a plasmid vector having a replication initiation region different from that of the first plasmid vector and coexisting in a microorganism together with the vector, for example, a plasmid pAMα1 derived from Streptococcus faecalis (Clewill, DB et al., Pro.
c. Natl. Acad. Sci. USA, 72, 1720-2724, (1975) and p.
HY300PLK (Ishiwa, H and Tsucida, N., Gene,
32, 129 (1984)), Staphylococcus aureus (St
aphylococcus aureus) derived plasmid pC194 (G
ros, MF et al., EMBO J., 6, 3863-3869 (1987))
RCR format (Gros, MF et al., The EMBO Journa
1, 6, 3863-3869, (1987)).

The thus constructed first plasmid vector and at least one or more other plasmid vectors are introduced into a microorganism by electroporation or the like, and grown in the same host while coexisting. A recombinant can be obtained by causing homologous recombination (see FIG. 1).

The microorganism used here is preferably a prokaryote, and more preferably a bacterium belonging to the genus Bacillus from the viewpoint of safety and secretion of enzymes.

Thus, using the method of the present invention, a recombinant gene can be formed in a microorganism.
When the homologous recombination is used to produce a recombinant gene, the lower the homology between the material genes is, the lower the degree of homology between them is, theoretically, almost infinite species. It is considered that it is almost impossible to construct and reintroduce all into host cells. According to the method of the present invention, creation of a recombinant gene in a host body is repeated indefinitely without ending, and since a recombinant gene of interest is constructed and simultaneously appears in a transformant as an expression system, If a positive screening can be performed with the expression system as it is as a transformant, only the target gene can be preferentially obtained, which is effective. For example, when trying to obtain a novel antibiotic resistance gene, a gene having resistance ability is constructed and at the same time, it grows preferentially, which makes screening very easy.

EXAMPLES Next, the present invention will be described in more detail by way of examples.

Example 1 (1) Construction of plasmid vector pTS43TC constructed from a DNA fragment containing a replication initiation region of plasmid DNA derived from Bacillus sp. KSM-KP43: The replication initiation region of plasmid DNA derived from Bacillus sp. The contained DNA fragment was obtained as follows.
That is, 0.5% of the Bacillus sp.
Polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.), 0.5% yeast extract (manufactured by Difco), 0.1% KH ₂ PO ₄ , 0.02%
The cells were cultured with shaking at 30 ° C. for 12 hours in 100 mL of a medium composed of MgSO ₄ .7H ₂ O and 0.5% Na ₂ CO ₃ . Plasmid DNA was obtained from the cells collected by centrifugation according to the method of Birnboim and Doly (Nucl. Acids. Res, 7, 1513-1523 (1979)). About 10 ng of the plasmid,
10 μL of PCR buffer (with Boehringer Pwo DNA polymerase), 2 types of oligonucleotides (primer 3; 5′-GAATTCCTGCAAGAAAACGATTGTG-
3 ′ (SEQ ID NO: 3)), (Primer 4; 5′-AAGATG
AGCTATAAGTCTTGTTAC-3 '(SEQ ID NO: 4)) 20 pmo
l, and 4 types of nucleotides (A, T, G, C) 20 pm each
ol was added and adjusted to 100 μL with deionized water. 94 ° C
After 2 minutes of treatment, 94 ° C for 1 minute, 55 ° C for 1 minute, 72
The reaction for 2 minutes at 72 ° C. was repeated for 30 cycles.
After performing the reaction using mal Cycler (Perkin Elmer), a 2.2 kbp DNA fragment containing the replication initiation region of the Bacillus sp. KSM-KP43-derived plasmid DNA was transferred to The GENECLEAN kit (Bio 101).
And the ends were blunt-ended with T4 DNA polymerase.

Next, 1.6 kbp of a DNA fragment containing the tetracycline gene on the plasmid pAMα1 derived from Streptococcus faecalis was amplified by PCR. Using about 1 ng of a plasmid pAMα1 derived from Streptococcus faecalis as a template, a PCR buffer (with Boehringer Pwo DNA polymerase)
10 μL, two kinds of oligonucleotides (primer 1; 5′-TGCAATGTGGAATTGGGAACGG-3 ′ (SEQ ID NO: 5)), (primer 2; 5′-CCCTTAACGATTTAGAAATC
20 pmol of CC-3 '(SEQ ID NO: 6) and 20 pmol of each of the four nucleotides (A, T, G, C) were added, and the mixture was adjusted to 100 μL with deionized water. After the treatment at 94 ° C. for 2 minutes, the reaction was repeated for 30 cycles, with the reaction at 94 ° C. for 1 minute, 55 ° C. for 1 minute and 72 ° C. for 1 minute as one cycle.
The reaction at 2 ° C for 5 minutes was performed using DNA Thermal Cyc.
ler (Perkin Elmer), and then a 1.6 kbp DNA fragment containing the tetracycline gene was obtained.
After isolation using the GENECLEAN kit (manufactured by BIO 101) and blunting the ends with T4 DNA polymerase, the DNA fragment 2.2 containing the replication initiation region of the plasmid DNA derived from Bacillus sp. KSM-KP43 strain 2.2.
kbp and T4 DNA ligase. The Bacillus subtilis ISW1214 strain (leu A8, metB8, hsrM
Transformation of 1) was performed by the protoplast method (S. Chang and SN Ch
oen; DM3 medium for protoplast regeneration [0.5% sodium succinate (pH 7.3), 0.5%, containing 5 µg / mL tetracycline, according to Mol. Gen. Genet., 168, 111-115 (1978)). Casamino acid, 0.5% yeast extract, 0.35% K ₂ HPO ₄ , 0.15% KH ₂ P
O ₄ , 0.5% glucose, 20 mM MgCl ₂ ,
0.01% bovine serum albumin (manufactured by Sigma)].

The recombinant plasmid retained by the recombinant B. subtilis was prepared by the method of Birnboim and Doly (Nucl. Acids. Res, 7, 1513-
1523 (1979)) and the resulting recombinant plasmid was analyzed for restriction enzyme breakpoints using agarose gel electrophoresis to obtain plasmid vector pTS43TC.
(FIG. 2) was obtained.

(2) p
Kanamycin resistance gene derived from UB110 (kanamycin resistance enzyme consisting of 205 amino acids; Sadaie, Y et a
l., J. Bacteriol. 141, 1178-1182 (1980)).
The gene encoding the kanamycin resistance gene A in which the position of the 76th amino acid was deleted by 1 bp and the frame was shifted as a result was ligated to the pTS43TC constructed in the above (1) by a ligation reaction. Also,
The kanamycin resistance gene B in which the position of the 121st amino acid has been deleted by 1 bp and a frame shift has been made as a result.
Was ligated to a plasmid in which a chloramphenicol resistance gene was ligated to pAMα1 by a ligation reaction. Both of these plasmids were introduced into Bacillus sp. KSM-KP43 strain by electroporation. (Note that a transformant containing only the genes A and B has no kanamycin resistance and contains an LB agar medium containing 15 μg / mL kanamycin (Bactotryptone 1).
%, Sodium chloride 1%, yeast extract 0.5%, sodium carbonate 0.05%, agar 1.5%).

Next, the transformants containing both plasmids were placed on an LB agar medium (1% bactotryptone, 1% sodium chloride, 0.5% yeast extract, 0.5% sodium carbonate).
0.05%, agar 1.5%, tetracycline 10
(g / mL, chloramphenicol 5 μg / mL) for 2 days. The grown cells were collected, diluted 1000-fold, and grown again on the same LB agar medium for 2 days. Subsequently, the grown cells were collected and diluted 100-fold, then plated on an LB agar medium containing 15 μg / mL of kanamycin, and incubated for 2 days. As a result, several hundred transformants appeared. A plasmid was prepared from the resulting transformant, and the sequence was analyzed. As a result, a gene encoding a protein capable of exhibiting resistance to kanamycin was detected.

Example 2 A gene encoding amino acids 1 to 178 of a kanamycin resistance protein and a kanamycin resistance gene C (about 800 bases) containing a regulatory region of the kanamycin resistance gene were ligated in Example 1 (1). And ligated with pTS43TC constructed in. In addition, a kanamycin resistance gene D (about 650 bases) containing a stop codon from the 102nd amino acid was p-ligated by ligation reaction.
Ligation with C194 plasmid. Both of these plasmids were transformed into Bacillus subtilis by electroporation.
(The transformant containing only the C and D genes had no kanamycin resistance and 10 μl
g / mL kanamycin-containing LB agar medium (1% bactotryptone, 1% sodium chloride, 0.5% yeast extract, 1.5% agar) was confirmed in advance to be unable to grow). Next, transformants containing both plasmids were transformed into LB liquid medium (1% bactotryptone, 1% sodium chloride, 0.5% yeast extract, 7.5 μg / ml tetracycline, chloramphenicol
5 μg / mL) for 1 day, and 10 μL of the culture solution is plated on an LB agar medium containing 15 μg / mL of kanamycin.
Incubation was performed for one day. As a result, several hundred transformants appeared. A plasmid was prepared from the resulting transformant, and the sequence was analyzed. As a result, a gene encoding a protein capable of exhibiting resistance to kanamycin was detected.

[0026]

According to the method for forming a recombinant gene of the present invention, the function of a recombinant gene can be evaluated in a microorganism as it is,
It is possible to directly search for the target gene. That is, the target gene can be searched for without performing an extra operation of returning the recombinant gene into the microorganism one by one as in the conventional gene recombination operation.

[0027]

[Sequence List] SEQUENCE LISTING <110> KAO CORPORATION <120> Method of forming recombinant genes <130> P00181301 <160> 6 <210> 1 <211> 2263 <212> DNA <213> Bacillus sp. KSM-KP43 <400 > 1 agatgagcta taagtcttgt tacggttccg aacccaaccc gtttgatgaa cacataatga 60 atttgaagga attagcagtt gaattaggaa acactgaagt gattactgtt attgcagact 120 attttaagtc cgatataaca gggttaacca aatctcagag gaagacctta aagaagtttc 180 tggacttgaa atattgggag aaacatactc ctgttaagta gtatatgtta agtggtacat 240 gttatatggt atatgttaag tggtatgata tacacacttg aatctattga tttcatacca 300 ctcaataaag attttattat ttaaaaagaa ttagagaaat tataaacatc cttcttcttt 360 gtcctttttt gaaaaacaaa aaggactttt ctgttttcaa tatcatcttg gaatttgttt 420 tttcgacaga caatagcacc agatacaaaa ttctgatagt attatagaca aagaaaaaac 480 tccctcaagg tttggcggcc aacgggagtt cttgtgcaag ggattagtgt acccttttga 540 cattgtattt aatttattta taccacaaat atatcatagg tacacgtccc tttccactat 600 gaaaggggaa aaaataatgg aaaatgcaaa ctccattcag aaggcgcaat atcacgcctg 660 gttccaacat agtgatgctg atggatggat tacagtagca agaaaaggtg agagtaatag 720 cttcatccag aaacactata agcctactga acttgcggac aagctgacag aatggttagg 780 agaagatgta ttctttagcc aaaatacatt ttatcggcca caaagaagca tagagaacgt 840 ccggcaatta cggtccttat atactgactt ggatttttat ttatttaact acgatccatc 900 ctgggtgatg gggaaactgc agcatgaatt ttttggccaa tcgataccag aaccgaactt 960 aattattttt agtggccaag gaatcgttct gatatggttg ttggatcctg tacctcataa 1020 agctctaccc ttatggcaag ctgtgcaaaa tcatttctta agccaattag aagagcttgg 1080 tggagatccg aaagctgctg atgctgcacg agtattccgt attgctggta gtgttaattc 1140 taagaacggg gcagaagtga gagcagagtt ccgtcatgat cataagtacc aacttaggca 1200 gctgcaatat gattacttac cggagcttaa cgatgagata aatccaccta agaagaaaaa 1260 aagaggacgc aagaaacggg tagcgcagtt atttaataca tacaaactgc attacgcacg 1320 tctgatggat ttagtgagat tggtagagtt gagaaactac caggtgaagg gtcatagaga 1380 gttaatctgc tttttatacc gatattggca atgttgctat tcaaacgatc cgtcagaagc 1440 cctgaatcag accatgacgc ttaatttgca gttttcggca cccttaaccc tccgagaagt 1500 ggaaagagcc accagaagcg ccgagaaagc ttgggaagct cgtaacaacg aagaagctaa 1560 taaaattgca atcgataaag gttatccagg agccggctat aatattagca ataaaaagct 1620 aatttcttgg cttgatatct cttccgatga aatgacgcat ttatcgacca ttatagacgg 1680 tttagagaag aacagaagga aacgtattgc caacatggaa atgcgccgag aacagggcgt 1740 gaagcctcgt gaagagtata taaaagagca acatgctaaa acagataacc aattaacgaa 1800 cctgcagcaa gcattgcaag aaaatccgaa ggcaaaacgg aaggatttag ctgctatatt 1860 aggtgtttct gtgtttcgta ttgatcagtt aaaacgacaa ttaaaaagtt tgtagtggtc 1920 tgtacgctta tattatgggc gttagcctgt gggtttaatt tgaggtcttt tcgggggcct 1980 cttttgcttt tcttctgggg gaggatcctg gcgttacatc atgttccttt tgtatcgaga 2040 agttattccg tgcttattct actttacttt tcctgagcta cttccaaaat acggagattt 2100 taggtgattt atatactgtt tttataaatt tgtctccaaa aagtatattt catatgttat 2160 aattaaaagt tttaaaaaat atattgaccg aacatatgtt agtgttttat aattaaggta 2220 ggtcattaat acaactccca caatcgtttt cttgcaggaa ttc 2263 <210> 2 <211> 432 <212> PRT <213> Bacillus sp.KSM-KP43 <400> 2 Met Glu Asn Ala Asn Ser Ile Gln Lys Ala Gln Tyr His Ala Trp Phe 5 10 15 Gln His Ser Asp Ala Asp Gly T rp Ile Thr Val Ala Arg Lys Gly Glu 20 25 30 Ser Asn Ser Phe Ile Gln Lys His Tyr Lys Pro Thr Glu Leu Ala Asp 35 40 45 Lys Leu Thr Glu Trp Leu Gly Glu Asp Val Phe Phe Ser Gln Asn Thr 50 55 60 Phe Tyr Arg Pro Gln Arg Ser Ile Glu Asn Val Arg Gln Leu Arg Ser 65 70 75 80 Leu Tyr Thr Asp Leu Asp Phe Tyr Leu Phe Asn Tyr Asp Pro Ser Trp 85 90 95 Val Met Gly Lys Leu Gln His Glu Phe Phe Gly Gln Ser Ile Pro Glu 100 105 110 Pro Asn Leu Ile Ile Phe Ser Gly Gln Gly Ile Val Leu Ile Trp Leu 115 120 125 Leu Asp Pro Val Pro His Lys Ala Leu Pro Leu Trp Gln Ala Val Gln 130 135 140 Asn His Phe Leu Ser Gln Leu Glu Glu Leu Gly Gly Asp Pro Lys Ala 145 150 155 160 Ala Asp Ala Ala Arg Val Phe Arg Ile Ala Gly Ser Val Asn Ser Lys 165 170 175 Asn Gly Ala Glu Val Arg Ala Glu Phe Arg His Asp His Lys Tyr Gln 180 185 190 Leu Arg Gln Leu Gln Tyr Asp Tyr Leu Pro Glu Leu Asn Asp Glu Ile 195 200 205 Asn Pro Pro Lys Lys Lys Lys Arg Gly Arg Lys Lys Arg Val Ala Gln 210 215 220 Leu Phe Asn Thr Tyr Lys Leu His Tyr Ala Arg Leu M et Asp Leu Val 225 230 235 240 Arg Leu Val Glu Leu Arg Asn Tyr Gln Val Lys Gly His Arg Glu Leu 245 250 255 Ile Cys Phe Leu Tyr Arg Tyr Trp Gln Cys Cys Tyr Ser Asn Asp Pro 260 265 270 Ser Glu Ala Leu Asn Gln Thr Met Thr Leu Asn Leu Gln Phe Ser Ala 275 280 285 Pro Leu Thr Leu Arg Glu Val Glu Arg Ala Thr Arg Ser Ala Glu Lys 290 295 300 Ala Trp Glu Ala Arg Asn Asn Glu Glu Ala Asn Lys Ile Ala Ile Asp 305 310 315 320 Lys Gly Tyr Pro Gly Ala Gly Tyr Asn Ile Ser Asn Lys Lys Leu Ile 325 330 335 Ser Trp Leu Asp Ile Ser Ser Asp Glu Met Thr His Leu Ser Thr Ile 340 345 350 350 Ile Asp Gly Leu Glu Lys Asn Arg Arg Lys Arg Ile Ala Asn Met Glu 355 360 365 Met Arg Arg Glu Gln Gly Val Lys Pro Arg Glu Glu Tyr Ile Lys Glu 370 375 380 Gln His Ala Lys Thr Asp Asn Gln Leu Thr Asn Leu Gln Gln Ala Leu 385 390 395 395 400 Gln Glu Asn Pro Lys Ala Lys Arg Lys Asp Leu Ala Ala Ile Leu Gly 405 410 415 Val Ser Val Phe Arg Ile Asp Gln Leu Lys Arg Gln Leu Lys Ser Leu 420 425 430 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <400> 3 gaattcctgc aagaaaacga ttgtg 25 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <400> 4 aagatgagct ataagtcttg ttac 24 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <400> 5 tgcaatgtgg aattgggaac gg 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <400> 6 cccttaacga tttagaaatc cc 22

[Brief description of the drawings]

FIG. 1 is a conceptual diagram showing a method for forming a recombinant gene of the present invention.

FIG. 2 is a view showing a process of constructing a plasmid vector pTS43TC.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Katsuhisa Saeki 2606 Akabane, Kaigamachi, Haga-gun, Tochigi Prefecture F-term in the Kao Corporation Research Laboratories (reference) 4B024 AA20 BA80 CA01 DA07 FA01 GA14 HA12

Claims (2)

[Claims] 1. A method for recombining two or more homologous genes in the same microorganism, wherein one gene lacks one or more nucleotides in the nucleotide sequence shown in SEQ ID NO: 1. A first plasmid comprising a lost, substituted or added nucleotide sequence or an amino acid sequence represented by SEQ ID NO: 2 or a nucleotide sequence encoding an amino acid sequence in which one or more amino acids have been deleted, substituted or added to the amino acid sequence A method for forming a recombinant gene in which a vector and another plasmid vector in which another gene is linked to a replication initiation region different from the first plasmid vector are introduced into a microorganism, and homologous recombination between the genes is caused. 2. The method according to claim 1, wherein the microorganism is a Bacillus bacterium.