JP6176597B2 - Neisseria gonorrhoeae with low TCA production ability and method for producing the same - Google Patents

Description

  The present invention relates to a method for producing koji mold having a reduced ability to produce 2,4,6-trichloroanisole (TCA) that causes musty odor in sake.

  The musty odor is an important issue because it significantly impairs the quality of sake. The causative substance is mainly 2,4,6-trichloroanisole (TCA), and the TCA contamination route is the conversion of 2,4,6-trichlorophenol (TCP) to TCA by koji mold in the sake brewing process. In addition, it has been reported that TCA contamination is caused from the environment such as wooden pallets during sake storage (Non-Patent Documents 1 to 3). The recognition threshold of TCA is 1.7 ng / l for sake (Non-Patent Document 4) and 2 ng / l for wine (Non-Patent Document 5), which is extremely low. On the other hand, TCP remains on wood and cork sprayed as an insect and fungicide, and lignin in timber is decomposed and chlorinated by chlorinated fungicides such as sodium hypochlorite. Is known (Non-Patent Document 2).

  In Aspergillus oryzae, it is confirmed that all seed potatoes have TCP methylation ability in tests using commercially available seed potatoes, and that TCA is generated by cocoon making in the presence of TCP (Non-patent Document 3). The enzyme involved in the conversion of TCA to TCA has not been identified. In order to prevent mold odor, it is necessary to use wooden tools that do not contain TCP, and not to use chloric acid-based disinfectants. Therefore, the breeding of Aspergillus oryzae that does not have the ability to methylate TCP is desired.

J. Biosci. Bioeng., 100, 178-183 (2005) Brewery, 102, 90-97 (2007) Brewery, 104, 777-786 (2009) Brewery, 99, 652-658 (2004) J. Agric. Food Chem., 50, 1032-1039 (2002)

  An object of the present invention is to identify a gene encoding an enzyme involved in the conversion of TCP to TCA in Neisseria gonorrhoeae, and to provide a novel means capable of preventing the production of TCA, which is a major causative substance of the musty odor of sake. It is to provide.

  The inventors of the present application extracted a number of genes presumed to encode methyltransferases from the Aspergillus oryzae RIB40 genome database (http://nribf2.nrib.go.jp/). Sequentially destroy and make gene-disrupted strains earnestly, perform iron making under the condition that TCP was added using each disrupted strain, and measure the TCA content in the obtained sputum, so that AO080521000231 gene-disrupted strain and parent strain As a result, it was found that the TCA content in sputum was significantly reduced, and the present invention was completed.

That is, the present invention relates to a method for producing a koji mold having reduced 2,4,6-trichloroanisole production ability, comprising reducing or deleting the expression of the AO080521000231 gene in koji mold, wherein the AO080521000231 gene has the sequence number 2 Or the amino acid sequence in which one to several amino acids are substituted, deleted, inserted or added in the sequence, and the genome sequence is 2001nt- in the nucleotide sequence shown in SEQ ID NO: 3. A method is provided which is a gene having 90% identity or more with 3460 nt . The present invention also encodes the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence in which one to several amino acids are substituted, deleted, inserted or added in the sequence, and the genomic sequence is SEQ ID NO: Provided a gonococci with low 2,4,6-trichloroanisole-producing ability in which the expression of the AO080521000231 gene, which is 90% or more identical to 2001nt-3460nt in the nucleotide sequence shown in 3, is reduced or deleted To do. Furthermore, the present invention relates to an upstream homologous region consisting of a region homologous to a continuous region of 500 bases or more in the 1 nt-2000 nt region in the base sequence shown in SEQ ID NO: 3, and in the base sequence shown in SEQ ID NO: 3. 3461nt-5460nt, a DNA fragment containing a region of 500 bases or more and a downstream homologous region consisting of a homologous region, and does not contain a normal AO080521000231 gene, and the AO080521000231 gene is shown in SEQ ID NO: 2 An amino acid sequence, or an amino acid sequence in which one to several amino acids are substituted, deleted, inserted or added in the sequence, and the genome sequence is 2001nt-3460nt in the nucleotide sequence shown in SEQ ID NO: 3 Provided is a DNA fragment , which is a gene having 90% or more identity . Furthermore, the present invention provides a method for producing sake having a low 2,4,6-trichloroanisole content, which comprises producing sake using the koji propagated with the koji mold of the present invention as a raw material.

  According to the present invention, the koji mold that encodes the major O-methyltransferase involved in the conversion of TCP to TCA is identified for the first time in the koji making process, and koji mold that suppresses TCA production during koji making even in the presence of TCP is the first time. offered. By producing koji using the koji mold of the present invention and producing sake using this koji as a raw material, even if using wooden utensils that may contain TCP, the production of TCA during koji making will be greatly increased. Since it can be suppressed, sake with a low TCA content and no moldy odor can be produced.

It is a figure explaining the outline of fusion PCR for amplification of the DNA fragment for gene destruction. A, B, C, D, A2, D2, adeA-F, and adeA-R are PCR primers. It is a graph which shows the relative value of the TCA content of the koji produced in the presence of TCP. It is a figure explaining the detail of PCR performed for confirmation of gene destruction.

  The inventors of the present application have identified the AO080521000231 gene for the first time as a gene encoding a major enzyme responsible for the reaction of methylating TCP in the koji making process and converting it into TCA. This gene is located on chromosome 8 of the Neisseria gonorrhoeae genome and is known to encode an S-adenosylmethionine (SAM) -dependent methyltransferase. The Aspergillus oryzae RIB40 genome database (http : //nribf2.nrib.go.jp/) is registered as AO080521000231. SEQ ID NOs: 1 and 2 show the cDNA sequence of the AO080521000231 gene registered in the database and the amino acid sequence of the protein encoded thereby. The AO080521000231 gene and the genomic sequence in the vicinity thereof are shown in SEQ ID NO: 3. The region of 2001nt-3460nt in SEQ ID NO: 3 is the coding region of the AO080521000231 gene.

  In the present invention, the “AO080521000231 gene” is not limited to a gene encoding a protein having the same sequence as the amino acid sequence shown in SEQ ID NO: 2 and having a genome sequence identical to 2001nt-3460nt of SEQ ID NO: 3, AO080521000231 gene encoding a protein consisting of a mutated sequence containing a mutation that can occur in the above and having a methyltransferase activity is also included. Such mutated sequences usually comprise a few (eg, 1-20, 1-15, 1-10, or 1 to several) bases or amino acid substitutions, deletions, insertions or additions, Although not particularly limited, the identity with the original sequence is 90% or more, for example, 95% or more, or 98% or more. Here, in the case of amino acid sequences, the “identity” of the sequences means that both amino acid sequences are aligned so that the amino acid residues of the two amino acid sequences to be compared match as much as possible. The number divided by the total number of amino acid residues is expressed as a percentage. In the above alignment, a gap is appropriately inserted in one or both of the two sequences to be compared as necessary. Such sequence alignment can be performed using a known program such as BLAST, FASTA, CLUSTAL W, and the like. When gaps are inserted, the total number of amino acid residues is the number of residues obtained by counting one gap as one amino acid residue. When the total number of amino acid residues counted in this way is different between the two sequences to be compared, the identity (%) is the total number of amino acid residues in the longer sequence, and the number of amino acid residues matched. Is calculated by dividing. The same applies to the identity of the base sequence.

  In the present invention, the expression of the AO080521000231 gene is reduced or deleted in Neisseria gonorrhoeae. Genetic modification methods for reducing or deleting the expression of a specific gene are widely known in this field, and can be appropriately selected and executed by those skilled in the art. Specific examples include, but are not limited to, antisense method, RNAi, gene disruption method and the like. In the present invention, it is preferable to disrupt the AO080521000231 gene.

  “Disrupting the AO080521000231 gene” means that the coding region of the AO080521000231 gene on the gonococcal genome is deleted or a normal gene product cannot be produced in at least some alleles of the gonococcal genome, preferably all alleles. It means to mutate. In the deletion of the AO080521000231 gene, the entire coding region may be deleted, or a part thereof may be deleted. When deleting the entire region, the region adjacent to the AO080521000231 gene may also be deleted widely. When a part is deleted, it is not particularly limited, but it is preferable to delete more than half of the coding region of the AO080521000231 gene. When the AO080521000231 gene is disrupted by a mutation in the coding region, for example, a nonsense mutation or a frameshift mutation is preferably introduced upstream of the center of the coding region, and a truncated protein having no methyltransferase activity Or it can be a completely unrelated protein sequence. A part or all of the AO080521000231 gene may be replaced with another sequence (for example, a marker gene sequence).

  Methods for gene disruption of Aspergillus oryzae are known, and the target gene can be disrupted by a technique using homologous recombination. For example, Katsuhiko Kitamoto, Junichi Maruyama: Latest technology and functionality of fermented and brewed foods (CMC Publishing) , Tokyo), 95-103 (2006). Specifically, for example, the upstream and downstream genomic regions of the AO080521000231 gene are amplified from the koji mold genome by PCR to prepare the upstream homologous region and the downstream homologous region, and at both ends of the sequence not containing the normal AO080521000231 gene. Two homologous regions may be connected to prepare a DNA fragment for gene disruption, and this may be introduced into Neisseria gonorrhoeae cells. By the action of the endogenous enzyme of Aspergillus oryzae, homologous recombination occurs between homologous regions, and the AO080521000231 gene region in the genome is replaced with a DNA fragment for gene disruption, so that the AO080521000231 gene can be deleted from the genome.

  As a homologous region, a fragment consisting of the same base sequence as the corresponding region in the gonococcal genome is usually used. However, even if the sequence is partially different from the corresponding region, homologous recombination occurs in the gonococcal cell. Can be used as a homologous region. That is, the homologous region includes the same base sequence as the corresponding portion in the base sequence shown in SEQ ID NO: 3, and a small number (preferably one or several) to the extent that homologous recombination occurs in the base sequence. ) Are substituted, deleted, inserted, or added. The identity between the homologous region and the corresponding region in SEQ ID NO: 3 is 90% or more, preferably 95% or more, more preferably 98% or more, and most preferably 100%. In the present invention, a “homology region” with a certain region refers to a region comprising a base sequence having the same identity as that region as described above.

  Although the size of the homologous region is not particularly limited, the longer the chain length, the higher the efficiency of homologous recombination, and if it becomes too short, the efficiency may decrease, so it is usually 30 bases or more. Those having about 500 bases or more, preferably about 700 bases or more are preferably used. The upper limit of the size is not particularly limited, but is usually 10,000 bases or less, preferably 2500 bases or less for the convenience of DNA synthesis.

  For example, in the sequence shown in SEQ ID NO: 3, since the AO080521000231 gene coding region is 2001nt-3460nt, a region of 500 bases or more, preferably 700 bases or more of the upstream region of 1nt-2000nt is amplified to form upstream homology. A region of 500 bases or more, preferably 700 bases or more in the downstream region of 3461nt-5460nt can be amplified to obtain downstream homologous regions. In the following examples, 723nt-1535nt in SEQ ID NO: 3 is amplified to form an upstream homologous region, and 3521nt-4947nt is amplified to form a downstream homologous region. However, the present invention is not limited thereto. SEQ ID NO: 3 shows the sequence of 2000 bases upstream and downstream of the AO080521000231 gene coding region, and further upstream and downstream sequences can be obtained from the Aspergillus oryzae RIB40 genome database described above. .

  The “normal AO080521000231 gene” is a gene that encodes a protein that can normally exhibit methyltransferase activity that converts TCP to TCA in the koji making process. The “sequence not including the normal AO080521000231 gene” linked to the homologous region may be a gene sequence other than the AO080521000231 gene, or may be a mutant AO080521000231 gene that does not encode a protein that functions as a normal methyltransferase. By using a marker gene as another gene sequence, screening of transformants (gene-disrupted strains) is facilitated.

  As the marker gene, an auxotrophic complementary gene suitable for the auxotrophy of the strain to be introduced, various drug resistance genes and the like can be appropriately selected and used. In the case of an auxotrophic marker, it is necessary to use a strain originally lacking the corresponding marker gene as the parent strain for gene disruption. From the viewpoint of obtaining, an auxotrophic marker gene is advantageous. On the other hand, the drug resistance marker is advantageous because it has no limitation on the genetic background of the parent strain to be used and can be used against a wider range of koji molds.

  Various marker genes are known and can be appropriately selected and used. For example, as an auxotrophic marker gene, adeA gene (involved in adenine synthesis and complementary to adenine requirement), sC gene (involved in sulfate (sulfur source) utilization, and sulfate (sulfur source) utilization-deficient strains Nutrient requirement complementation), argB gene (which is involved in arginine synthesis and complements arginine requirement), etc. are known. The sequences of these genes are known, and sequence information can be obtained from the Aspergillus oryzae RIB40 genome database described above, NCBI GenBank, and the like. For example, in the following examples, a strain that lacks adeA and is adenine-requiring is used as a parental for gene disruption, and adeA gene is used as a marker gene to screen a gene-disrupted strain using adenine-requiring as an index. Yes. The sequence of the adeA gene is registered as AO080527000383 in the Aspergillus oryzae RIB40 genome database and with an accession number of AB121755 in GenBank. The sequence shown in SEQ ID NO: 4 is the adeA gene and the genomic sequence in the vicinity thereof. In the following Examples, a DNA fragment for gene disruption is prepared using a region of 360nt-2362nt, but is not limited thereto. Examples of the drug resistance marker gene include, but are not limited to, the ptrA gene (resistance gene for pyrithiamine which is an antimetabolite analog of thiamine).

  The upstream homologous region and the downstream homologous region can be linked to the marker gene DNA by the fusion PCR method (see FIG. 1). When the upstream homologous region and the downstream homologous region are amplified from the genome, a complementary region that hybridizes with a partial region at the end of the marker gene is added to one of the primer pairs (B and C in FIG. 1). When three fragments are hybridized and fused to a single DNA fragment and PCR is performed using this fragment as a template, the [upstream homologous region]-[marker gene]-[downstream homologous region] are linked. A DNA fragment for gene disruption can be amplified. The direction of the marker gene in the DNA fragment may be the same as or opposite to that in FIG.

  The DNA fragment for gene disruption may be incorporated into a plasmid vector such as pUSC and introduced into gonococcal cells (Bioscience, Biotechnology, and Biochemistry, 61 (8), 1367-1369, 1997), or in the examples below. As described above, the DNA fragment can also be introduced into a koji mold cell by the protoplast method. Briefly describing the protoplast method, the conidia of Aspergillus oryzae are cultured in a liquid medium for about 24 hours, and then filtered through a non-woven fabric or the like having a pore size of about 20 to 30 μm to recover the bacterial cells. It is possible to obtain Neisseria gonorrhoeae protoplasts by culturing for several hours in a protoplasting solution containing chitinase, chitobiase, β-1,3-glucanase, etc.) and then removing the cell residue by filtration through a glass filter or the like. it can. The protoplast suspension and the gene disruption DNA are mixed, ice-cooled for several tens of minutes, mixed with polyethylene glycol, allowed to stand, and then cultured on an agar selective medium.

  Transformants obtained after screening with a marker are homozygous strains in which all alleles of AO080521000231 gene are disrupted, or some alleles of AO080521000231 genes are disrupted and at least one allele of AO080521000231 genes is not disrupted. Although it is a remaining hetero-disrupted strain, an AO080521000231 non-disrupted strain in which a DNA fragment for gene disruption is ectopically integrated may be obtained. Therefore, it is desirable to carry out Southern analysis, PCR amplification product confirmation, direct sequencing, etc. as appropriate after screening with a marker to directly confirm that the DNA fragment is inserted at the intended position and the AO080521000231 gene is destroyed.

  In the present invention, a homo-disrupted strain that can completely delete the expression of the AO080521000231 gene is preferable. Homo-disrupted strains can be obtained directly by the gene disruption method described above, and the obtained hetero-disrupted strain is used as the parent strain for gene disruption, and gene disruption treatment is repeated until all allele AO080521000231 genes are disrupted. May be. If a different marker gene is used for each gene disruption treatment, screening using the marker gene becomes possible. In addition, when a gene-disrupted strain is prepared using a mating gonococcal strain, a homo-disrupted strain can also be obtained by mating heterozygous strains.

  In the present invention, the kind of Aspergillus is not particularly limited, and widely includes Aspergillus genus bacteria such as Aspergillus oryzae, Aspergillus niger, Aspergillus awamori, and Aspergillus shirousamii, Aspergillus kawachii. To do.

  In the koji mold in which the expression of the AO080521000231 gene is reduced or deleted, the ability to produce TCA is reduced. That is, the ability to generate TCA from TCP is lower than that of the original parent strain having the normal AO080521000231 gene. In a koji strain in which the gene has been disrupted, the ability to produce TCA can be reduced to less than 20% of the parent strain, such as less than 15%. Even if brewing in the presence of TCP, the amount of TCA produced is low, so if the koji mold of the present invention is propagated to rice etc. to produce koji, and if sake is made from this koji, TCP may be contained Even using wooden utensils, etc., can significantly reduce the production of TCA and produce sake with a low TCA content. The recognition threshold of TCA is as low as 1.7 ng / l for sake. Generation of mold odor can be easily prevented.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

1. Creation of Neisseria gonorrhoeae gene disruption strain
Aspergillus oryzae NSR-ΔlD2 strain (niaD ^- sC ^- adeA ^- ΔligD) (Maruyama, J., Kitamoto, K .: Biotechnol. Lett., 30,1811-1817 (2008)) as a host, and the adeA gene (AO080527000383) The AO080521000231 gene was disrupted as a marker. The fusion PCR method and the protoplast method were performed according to the method described in Katsuhiko Kitamoto and Junichi Maruyama: Latest Technology and Functionality of Fermented and Brewed Foods (CM Publishing, Tokyo), 95-103 (2006).

(1) Preparation of DNA fragment for AO080521000231 gene disruption First, genomic DNA was extracted from Aspergillus oryzae RIB40 strain using DNeasy Plant Maxi Kit manufactured by QIAGEN. Using this as a template, a DNA fragment was prepared by the fusion PCR method (FIG. 1). As a first step, a region adjacent to the 5 ′ side of the target AO080521000231 gene (“upstream homologous region”, 723nt-1535nt in SEQ ID NO: 3, primers used are A and B) and a region adjacent to the 3 ′ side ("Downstream homologous region", 3521nt-4947nt in SEQ ID NO: 3, primers used are C and D) and adeA gene region (adeA fragment, 360nt-2362nt in SEQ ID NO: 4, primers used are adeA-F and adeA- A total of three fragments (R) were amplified by conventional PCR. Complementary sequences to the 5 ′ end portion and 3 ′ end portion of the adeA fragment were respectively added to the 5 ′ primer B and the 3 ′ primer C in the target gene amplification (lower case portion in Table 1).

  Subsequently, these three fragments were fused by fusion PCR to prepare a DNA fragment for destruction in which the target gene was replaced with the adeA gene. KOD-Plus-Ver.2 manufactured by Toyobo Co., Ltd. was used as the PCR enzyme, and fusion PCR was 94 ° C for 2 minutes (94 ° C for 15 seconds, 64 ° C for 30 seconds, 68 ° C for 3 minutes) x 30 cycles, 68 It was performed under the condition of 2 ° C. Finally, fusion PCR products were purified and concentrated (concentration 0.2 to 2 μg / μl) using QIAGEN QIAquick PCR Purification Kit.

(2) Transformation The prepared DNA fragment was introduced into Aspergillus oryzae NSR-ΔlD2 strain by the protoplast method. For the preparation of protoplasts, 200 ml of medium (glucose 4 g, bactotryptone 0.2 g, yeast extract 1 g, NaNO ₃ 0.2 g, K ₂ HPO ₄ 0.1 g, MgSO ₄ · 7H ₂ O 0.1 g, FeSO ₄ · 7H ₂ O 0.002 g and adenine 0.1 g (pH 6.0)) were inoculated with about 1 × 10 ⁸ conidia of NSR-ΔlD2 strain and cultured with shaking at 30 ° C. and 105 rpm for 24 hours. The culture solution is filtered through CALBIOCHEM Miracloth to recover the cells, and 20 ml of protoplast solution (0.5% Yatalase (Takara Bio), 0.8 M NaCl, 10 mM phosphate buffer (pH 6.0)) It was transferred to a centrifuge tube containing, and shaken at 30 ° C and 80 rpm for 3 hours. Then, it filtered with Miracloth and a glass filter (IWAKI 3G1) to remove the cell residue, and the filtrate was collected. The filtrate was centrifuged at 3000 rpm for 5 minutes at 4 ° C, and solution 1 (0.8 M NaCl, 10 mM CaCl ₂ , 10 mM Tris-HCl (pH 8.0)) was added to the precipitate at a protoplast concentration of 2.4 x 10 ⁸ cells / ml. And suspended.

For introduction of DNA fragments, first add 12.5 μl of Solution 2 (40% PEG4000, 50 mM CaCl ₂ , 10 mM Tris-HCl (pH 8.0)) and 5 μl of DNA fragment for disruption to 50 μl of protoplast suspension. And left on ice for 30 minutes. As controls, the ones to which the adeA gene (PCR product of adeA-F and adeA-R, FIG. 1) was added as a DNA fragment and the one without the DNA fragment were prepared in the same manner. Next, 500 μl of Solution 2 was added, mixed gently, and allowed to stand at room temperature for 15 minutes. Finally, 1 ml of Solution 1 was added and mixed gently. A portion of this was added to 2% agar selective medium (glucose 20 g, L-glutamic acid 3 g, KCl 0.5 g, NaCl 46.75 g, K ₂ HPO ₄ 1 g, MgSO ₄ · 7H ₂ O
After adding 0.5 g of FeSO ₄ · 7H ₂ O 0.02 g and L-methionine 1.5 g / l (pH 5.5)), 5% to 10 ml of 0.7% soft agar selective medium was added and overlaid. After culturing at 30 ° C for 1 week, colonies appeared and planted several times in selective medium to stabilize the character.

  For confirmation of gene disruption, the transformed strain on the agar medium was scraped and direct PCR was performed using KOD-FX manufactured by Toyobo Co., Ltd. according to the instruction manual. Three combinations of A2 and D2, adeA-F and D, and A and adeA-R (Table 1) were used as PCR primers (FIG. 3).

2. Measurement of TCP methylation ability of Aspergillus oryzae
(1) Steelmaking Weigh 15 g of α rice (milled rice ratio 70%) in a 9 cm diameter glass petri dish, dry heat sterilized at 95 ° C for 2 hours, let cool, and contain TCP (10 ng / ml) 0.05 7.5 ml of% Tween 80 aqueous solution was added, and about 1 × 10 ⁶ conidia were inoculated per 1 g of α-rice. This was placed in a constant temperature and humidity chamber (THE101FA manufactured by Advantech), and brewed for 44 hours at a temperature of 35 ° C and a humidity of 80% (constant) (Brakyo, 104, 777-786 (2009), and Breweries, 73 402-404 (1978)). The petri dish was sealed with vinyl tape to prevent scattering of TCP and TCA, and the lid was opened at 18, 23, and 27 hours to supply oxygen and stir.

(2) Measurement of TCA content of rice cake
For extraction of TCA, 1 g of 麹 was put into a vial, 9 ml of 50% ethanol aqueous solution was added, and then left at 4 ° C for 24 hours. Add 8 ml of pure water and 2 g of sodium chloride to 2 ml of this extract and stir at 30 ° C, 800 rpm for 60 minutes using a stirrer coated with polydimethylsiloxane (Twister manufactured by GERSTEL). Was adsorbed. After adsorption, the amount of TCA was measured by GC / MS according to the method reported previously (Beikyo, 104, 777-786 (2009)). The measurement was performed twice and the average value (per gram of 麹) was shown.

3. Measurement of enzyme activity and bacterial mass
(1) Steelmaking Weigh 100 g of α rice (milled rice ratio 70%) in a glass petri dish with a diameter of 15 cm, sterilize by dry heat at 95 ° C for 2 hours, cool down, and contain TCP (10 ng / ml) 0.05 43.3 ml of% Tween 80 aqueous solution was added, and about 1 × 10 ⁶ conidia were inoculated per 1 g of α-rice. This was placed in a thermo-hygrostat and smelted for 48 hours (Bukyo, 73, 402-404 (1978)). The constant temperature and humidity chamber is 32 ° C for 24 hours, 95% humidity for 24 hours, 34 ° C for 95 hours, 95%, 38 ° C for 90 to 35 hours, 90% for 35 to 38 hours, 42 ° C for 85 hours, 85% After 38 hours, the temperature was set to 42 ° C and 80%. When the setting was changed (24th, 30th, 35th and 38th hours), the petri dish lid was opened, and oxygen was supplied and stirred. The petri dish is covered with glass for 24 hours from the start of iron making, and covered with filter paper (Qualitative filter paper No. 2 diameter 285 mm, manufactured by Advantech) for 24 to 35 hours, and then covered with glass for 35 hours. After that, it was covered only with filter paper.

(2) Measurement of various enzyme activities and cell mass of koji Prepare koji man's α-amylase measurement kit, saccharification power, by preparing koji enzyme solution according to the National Tax Agency's prescribed analysis method “211-4-2 Preparation of enzyme solution”. It measured using the measurement kit and the acid carboxypeptidase measurement kit, respectively. Acidic protease was measured according to the standard analysis method for alcoholic beverage research institute ("111-10 acidic protease", http://www.nrib.go.jp/data/nribanalysis.htm). Moreover, the amount of microbial cells of the cocoon was measured using a kit for measuring the gonococcal amount manufactured by Kikkoman. The measurement was performed three times, and the average value (per 麹 g) was shown.

Results and Discussion Several strains of AO080521000231 gene disruption (homo and hetero disruptions, confirmed by PCR shown in FIG. 3) were obtained by transformation. Colonies on the selective medium showed the same morphology in the AO080521000231 gene-disrupted strain and the control strain (adeA gene fragment introduced).

  Several AO080521000231 gene-disrupted strains (homo-disrupted strains) obtained by the transformation were each made using several strains, a control strain, and a RIB40 strain for reference. In addition, as already reported, the addition of 0 to 6.7 ng of TCP per gram of α-rice was carried out, and the relationship between the amount of TCP added and the amount of TCA produced was examined. As the amount of TCA increased, the amount of TCA produced also increased (J. Biosci. Bioeng., 100, 178-183 (2005), Shukyo, 102, 90-97 (2007)). It added so that it might become 5 ng per g of alpha rice. As a result, all the strains showed normal-looking wrinkles, and there was no difference in the appearance of wrinkles depending on the strains used.

  The TCA content of each cocoon was measured, the values of several disrupted strains used were averaged, and the TCA content of the control strain (2.1 ng per gram) was evaluated as a relative value of 1. The TCA of the AO080521000231 gene-disrupted strain was Was reduced 0.12 times (FIG. 2). In addition, TCA was not detected when α rice to which TCP was added was not inoculated with conidia and the same koji making operation was performed.

  Next, using the AO080521000231 gene disruption strain, the control strain, and the RIB40 strain, and without newly adding TCP, and making a new koji under conditions of actual temperature and humidity, various enzyme activities and bacterial cells of koji The amount was measured. As a result, no clear difference was observed in α-amylase activity, glucoamylase activity, acid carboxypeptidase activity, acid protease activity, and cell mass (Table 2). Therefore, it was found that the AO080521000231 gene-disrupted strain had decreased methylation ability of TCP while maintaining the ability to make normal sputum.

  AO080521000231 corresponds to AoEST07474 and has been confirmed to be expressed in rice bran. This gene encodes a major O-methyltransferase involved in the conversion of TCP to TCA during koji making. The possibility was suggested. The gene disruption of AO080521000231 did not completely suppress the production of TCA, suggesting that other genes may be involved.

  Previously, the gene cmt1 encoding chlorophenol O-methyltransferase (CMT1) of Trichoderma longibrachiatum was reported (GenBank accession no. FN554867) (Fungal Genet. Biol., 47, 458-467 (2010)). Comparing the deduced amino acid sequence of CMT1 with that of AO080521000231, the homology (identity) was only 27%, suggesting that the properties of the gonococcal enzyme are different from those of CMT1.

Claims (8)

A method for producing a koji mold having reduced 2,4,6-trichloroanisole production ability, comprising reducing or deleting the expression of the AO080521000231 gene in koji mold, wherein the AO080521000231 gene has the amino acid sequence shown in SEQ ID NO: 2, or The sequence encodes an amino acid sequence in which one to several amino acids are substituted, deleted, inserted or added, and its genomic sequence is 2001nt-3460nt in the nucleotide sequence shown in SEQ ID NO: 3 and 90% or more. A method, which is a gene having identity.   The method of claim 1, comprising disrupting the AO080521000231 gene in the gonococcal genome.   An upstream homologous region consisting of a region homologous to the genomic partial region upstream from the coding region of the AO080521000231 gene, and a downstream homologous region consisting of a region homologous to the genomic partial region downstream from the coding region of the AO080521000231 gene, The method according to claim 2, wherein the AO080521000231 gene is disrupted by introducing a DNA fragment not containing a normal AO080521000231 gene into a gonococcal cell and causing homologous recombination between the DNA fragment and the gonococcal genome.   The upstream homologous region is composed of a region homologous to a continuous region of 500 bases or more in the 1 nt to 2000 nt region in the base sequence shown in SEQ ID NO: 3, and the downstream homologous region is a base shown in SEQ ID NO: 3 The method according to claim 3, comprising a region homologous to a continuous region of 500 bases or more in the 3461nt-5460nt region in the sequence. It encodes the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence in which one to several amino acids are substituted, deleted, inserted or added in the sequence, and its genomic sequence is in the base sequence shown in SEQ ID NO: 3 Aspergillus oryzae having low 2,4,6-trichloroanisole production ability, wherein the expression of the AO080521000231 gene, which is a gene having 90% or more identity with 2001nt-3460nt, is reduced or deleted.   The koji mold according to claim 5, which has a genome in which the AO080521000231 gene is disrupted. An upstream homologous region consisting of a region homologous to a continuous region of 500 bases or more in the 1nt-2000nt region in the base sequence shown in SEQ ID NO: 3, and a 3461nt-5460nt region in the base sequence shown in SEQ ID NO: 3 A DNA fragment containing a normal region of 500 bases or more and a downstream homologous region consisting of a homologous region and not including a normal AO080521000231 gene , wherein the AO080521000231 gene is the amino acid sequence shown in SEQ ID NO: 2, or the sequence In which 1 to several amino acids are substituted, deleted, inserted or added, and the genome sequence is 90% or more identical to 2001nt-3460nt in the nucleotide sequence shown in SEQ ID NO: 3 A DNA fragment, which is a gene having   A method for producing sake having a low 2,4,6-trichloroanisole content, comprising producing sake using the koji produced by breeding koji mold according to claim 5 or 6 as a raw material.