JP6261039B2 - Method for degrading polysaccharides using Aspergillus oryzae in which transcriptional control factors manR and xlnR are expressed in a double forced manner - Google Patents

Description

The present invention relates to a method for decomposing a polysaccharide using soy sauce koji molds in which transcriptional control factors manR and xlnR are expressed by double forced expression, and further to a method for producing soy sauce using the method.

Aspergillus sojae and Aspergillus oryzae are brewing microorganisms that have long been used in Japanese food production, and are both industrially very important filamentous fungi (for example, Non-patent document 1).

  Numerous enzymes produced by koji mold in soy sauce brewing play an important role in the degradation of raw materials. Among them, saccharide-hydrolyzing enzymes are thought to function to reduce moromi viscosity in soy sauce brewing and to facilitate compression by decomposing polysaccharide components such as cellulose, hemicellulose and pectin derived from raw materials. .

  Among saccharide-degrading enzymes, gonococcal enzymes such as carboxymethylcellulose saccharifying enzyme (CMCase), pectin liquefying enzyme (pectinase), pectin lyase, and β-glucanase are used to reduce the viscosity of miso and improve filterability in soy sauce brewing, and It is known to play an important role in improving the compressibility of soy sauce moromi (see, for example, Non-Patent Document 2).

  Regarding CMCase, it is considered that it participates in xyloglucan degradation of soybean cell wall, and this leads to improvement of squeezability (for example, see Non-Patent Document 2).

  As described above, the action of the saccharide-degrading enzymes in soy sauce brewing is a factor that greatly affects the decrease in moromi viscosity and the improvement in pressability. Therefore, when the function of the carbohydrate hydrolase group produced by Aspergillus oryzae is weak at the time of soy sauce brewing, there is a problem that the squeezability decreases due to an increase in moromi viscosity, and the amount of liquid juice that can be recovered from moromi decreases. Will occur.

  As a means for preventing this problem, the use of koji mold that has enhanced the production of saccharide-degrading enzymes by forcibly expressing cellulase such as CMCase and genes encoding hemicellulase and pectinase in soy sauce brewing Conceivable. However, there is a limit to forcibly expressing a plurality of saccharide-degrading enzyme genes at the same time to enhance the gonococcal enzyme. In particular, the two types of transfer control factors do not always exhibit an additive effect, and even a synergistic effect cannot be predicted by those skilled in the art.

In addition, since polysaccharides derived from raw materials have a complicated structure, a plurality of enzyme groups having different properties must act in cooperation for efficient degradation. For example, in order to efficiently decompose 4- O -methylglucuronoarabinoxylan, which is one of hemicelluloses, not only endoxylanase or β-xylosidase that degrades the main chain of xylan, but also α which is a side chain degrading enzyme -The action of a plurality of enzymes such as L-arabinofuranosidase, α-glucuronidase, acetyl xylan esterase is necessary (for example, see Non-Patent Document 3).

  Therefore, the method of comprehensively increasing the production of the target saccharide-degrading enzyme group by forcibly expressing the gene for the regulator of saccharide-degrading enzyme promotes the degradation of polysaccharides derived from the raw material. It is considered effective.

  So far, in Aspergillus oryzae, XlnR, which is a positive transcriptional regulator of the xylan hydrolase gene group and the cellulose hydrolase group, has been discovered (for example, Non-Patent Document 4, Non-Patent Document 5 and Patent Document). 1).

  In addition, in this strain, in recent years, ManR, which is a positive transcriptional control factor of mannan hydrolase gene group and cellulose hydrolase gene group (see, for example, Non-Patent Document 6, Non-Patent Document 7 and Patent Document 2), has been seen. Has been issued.

麹 学 5th edition edited by Hideya Murakami Soy Sauce Science and Technology, edited by Toshiro Tochikura Hemicellose and Hemicelles, Edited by M.M. P. Coughlan and G.C. P. Hazlewood Fungal Genet. Biol. 35, 157-169 (2002) FEBS Lett. 528, 279-282 (2002) Fungal Genet. Biol. ; 49, 987-995 (2012) Biosci. Biotechnol. Biochem. 77, 426-429 (2013)

JP 2001-095581 A WO2010 / 101129

The present invention uses gonococci forcibly expressing the transcriptional regulatory factors manR and xlnR at the same time, thereby increasing the activity of saccharide-degrading enzymes in the cocoon , enhancing the degradation of the polysaccharide, and thus reducing the moromi viscosity. An object is to provide a method for producing the soy sauce.

Conventionally, whether the effects of cellulase and hemicellulase production are additively or synergistically expressed when the transcriptional regulatory factors manR and xlnR are simultaneously expressed simultaneously is not limited to the soy sauce koji Aspergillus soya but the koji mold Aspergillus sojae There have been no studies on other Aspergillus fungi such as Oryzae and black mold Aspergillus niger.

Under such circumstances, the present inventors have found that the degradation of polysaccharides is enhanced by forcibly expressing transcription regulators manR gene derived from Aspergillus soya and xlnR in fungi. The present invention was completed by using the strain for soy sauce brewing.

That is, the present application provides the following inventions:
The first invention relates to a method of decomposing manR gene or a homologous gene thereof and xlnR gene or polysaccharide using transformant that were forced to express a homologous gene. More specifically, the following gene (a) ( manR gene or homologous gene thereof) and gene (b) ( xlnR gene or homologous gene thereof) are forcibly expressed.
(a) any of the following genes (a-1) to (a-5):
(a-1) a gene comprising the nucleotide sequence set forth in SEQ ID NO: 1,
(a-2) a gene encoding the amino acid sequence set forth in SEQ ID NO: 3,
(a-3) a gene encoding a carbohydrate hydrolase transcriptional regulatory factor, comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence set forth in SEQ ID NO: 1.
(a-4) a gene encoding a carbohydrate hydrolase transcriptional regulatory factor, comprising an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 3, or
(a-5) a gene encoding a carbohydrate hydrolase transcriptional regulatory factor consisting of an amino acid sequence in which one or several amino acids of SEQ ID NO: 3 are deleted, substituted and / or added; and
(b) any of the following genes (b-1) to (b-5):
(b-1) a gene comprising the base sequence described in SEQ ID NO: 2,
(b-2) a gene encoding the amino acid sequence set forth in SEQ ID NO: 4,
(b-3) a gene encoding a carbohydrate hydrolase transcriptional regulatory factor, comprising a nucleotide sequence having 90% or more identity with the gene consisting of the nucleotide sequence of SEQ ID NO: 2;
(b-4) a gene encoding a carbohydrate hydrolase transcriptional regulatory factor comprising an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 4, or
(b-5) A gene encoding a carbohydrate hydrolase transcriptional regulatory factor consisting of an amino acid sequence in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 4 have been deleted, substituted and / or added.

Each of the above genes is placed in a vector linked so as to be under the control of an appropriate forced expression promoter. For example, gene (a) is preferably under the control of a translation elongation factor promoter and gene (b) is preferably under the control of an alkaline protease promoter. After producing such a vector, the desired transformant ( manR - xlnR double forced expression strain) is produced by introducing it into a host. Examples of hosts include, but are not intended to be limited to, filamentous fungi, preferably Aspergillus sojae. The polysaccharide to be decomposed may be a polysaccharide derived from a soy sauce raw material such as soybean and wheat.

  The second invention relates to a method for producing soy sauce using the polysaccharide decomposition method.

  The third invention relates to a recombinant vector containing the genes (a) and (b).

  The fourth invention relates to a transformant containing the above recombinant vector.

By this invention, decomposition | disassembly of the polysaccharide in soy sauce raw material is accelerated | stimulated, and it can provide the soy sauce manufacturing method which reduced the moromi viscosity by extension. As a result, the amount of juice recovered by natural drool from moromi increases. Moreover, since the moromi viscosity has fallen, it is anticipated that the savory taste produced by this manufacturing method is improving. Whether or not the fungi in which the two transcriptional regulator genes manR and xlnR are expressed in two forced manners exhibit useful properties such as enhanced degradation of polysaccharides, reduced mash taste viscosity and improved squeezability in soy sauce brewing Was not known at all before the present application.

Aspergillus soja KP-del strain production process Method for removing adeA gene from Aspergillus soja KP-del strain Confirmation of adeA gene deletion region by DNA sequence analysis Confirmation result of auxotrophy of KPA-del-S2-5R strain PyrG restoration confirmation result of Aspergillus soja KP-del strain Structure of the cassette for forced expression of the xlnR gene and its location in the host Confirmation result by PCR of xlnR- OE (Δ pyrG ) strain Structure of the cassette for forced expression of the manR gene and its location in the host Confirmation result of manR - xlnR -OE strain by PCR Confirmation result of manR single forced expression strain by PCR Results of confirming restoration of pyrG gene of xlnR- OE (Δ pyrG ) strain by PCR Effects of manR - xlnR double forced expression on mannan hydrolase group production in Aspergillus sojae Effects of manR - xlnR double forced expression on xylan hydrolase group production in Aspergillus sojae Effects of manR - xlnR double forced expression on cellulose hydrolase group production in Aspergillus soja ManR- state of koji and moromi in soy sauce raw material short-term decomposition test using xlnR double forced expression strain Results of Miso Viscosity Measurement in Soy Sauce Raw Material Short-term Decomposition Test Using manR - xlnR Double Forced Expression Strains Result of measurement of the amount of recovered juice by natural dripping in the soy sauce raw material short-term decomposition test using man R- xlnR double forced expression strain Production of manR - xlnR double forced expression strain (MXOE-N2 strain) in Aspergillus soja NBRC4241 strain Soy sauce raw material short-term decomposition test results using MXOE-N2 strain Moromi state in soy sauce small-scale brewing test using MXOE-N2 strain Miso viscosity measurement results in a small-scale soy sauce brewing test using MXOE-N2 strain Result of measuring the amount of recovered juice by natural dripping in a small-scale soy sauce brewing test using MXOE-N2

The manR - xlnR double compulsory expression strain used in the present invention was prepared by isolating SEQ ID NO: 1 ( manR gene) and SEQ ID NO: 2 ( xlnR gene) and preparing a vector linked to an appropriate promoter. It can be obtained by introducing it into a filamentous fungus such as soy sauce koji. Using the transformant thus obtained, the present invention can be carried out by decomposing the polysaccharide and thus brewing soy sauce.

Instead of the manR gene consisting of the base sequence of SEQ ID NO: 1 and the xlnR gene consisting of the base sequence of SEQ ID NO: 2, homologous genes encoding a transcriptional regulatory factor of a carbohydrate hydrolase may be used. The homologous gene preferably has, for example, 90% or more, preferably 95% or more, more preferably 98% or more identity with each gene. As for the protein encoding each gene, the gene encoding the protein can be used as long as the homologous protein is a transcriptional control factor of carbohydrate hydrolase. As used herein, “identity” refers to all overlapping in an optimal alignment when two base or amino acid sequences are aligned using mathematical algorithms known in the art. It means the ratio (%) of the same base or amino acid residue to the base or all amino acid residues. Preferably, the algorithm can allow for the introduction of gaps in one or both of the sequences for optimal alignment.

Production of the manR - xlnR double forced expression strain of the present invention can be easily carried out by those skilled in the art based on the disclosure of the present specification. For example, Aspergillus soya, more specifically, Aspergillus soya NBRC 4239 strain (deposit organization: National Institute of Technology and Evaluation, Biotechnology Headquarters, deposit number ID: NBRC4239).

Examples of the carbohydrate hydrolase whose production is enhanced in the manR − xlnR double forced expression strain include enzymes that degrade polysaccharide components such as cellulose, hemicellulose, and pectins. In particular, the double forced expression strain of the present invention can enhance the production of cellulase and hemicellulase, and therefore can be suitably used for the degradation of cellulose and hemicellulose.

Method for producing transformant The recombinant vector of the present invention can be obtained by ligating the manR gene and the xlnR gene on an appropriate vector. Any vector can be used as long as the two transcriptional regulatory factors are forcibly expressed in the host to be transformed. For example, the vector is not limited to a chromosome integration type, but can be maintained in a filamentous fungus. Vectors such as plasmids can also be used.

  When a vector for forced expression is introduced onto a host chromosome, the mode of introduction is not limited as long as the gene loaded on the vector is correctly forcibly expressed. For example, when integrating a vector into a chromosome, a sequence corresponding to a region on the target chromosome may be added to both ends of the vector to be integrated, and integrated using homologous recombination. Alternatively, the vector may be randomly integrated on the host chromosome by utilizing the incorporation of foreign DNA into the host chromosome by non-homologous recombination.

  The vector may include a marker gene to allow selection of transformed cells.

Examples of the marker gene include genes that complement the auxotrophy of the host, such as adeA , pyrG , argB , trpC , niaD , sC , and resistance genes for drugs such as pyrithiamine and aureobasidin.

Examples of the promoter used for forced expression of the target transcription control factor include alkaline protease promoter ( alp promoter), translation elongation factor promoter ( tef1 promoter), α-amylase promoter, and the like.

  The transformant of the present invention can be obtained by transforming a host with a recombinant vector. The host is not particularly limited as long as it is a fungus suitable for polysaccharide degradation, and examples thereof include filamentous fungi that can be used for brewing soy sauce such as Aspergillus soya and Aspergillus oryzae.

  Transformation can be performed by a known method depending on the host. In the case of using a filamentous fungus, for example, Mol. Gen. Genet. 218: 99-104, 1989 can be used.

  EXAMPLES Hereinafter, although this invention is concretely demonstrated according to an Example, the technical scope of this invention is not restrict | limited at all by these description.

-Estimation of the manR and xlnR genes of Aspergillus sojae Aspergillus soja NBRC4239 strain, the results of its draft genome analysis was published in 2011 (DNA Research; 18, 165-176, 2011). The base sequence of the bacterial scaffold is registered and published as DDBJ / ENA / Genbank accession numbers DF093557 to DF093585.

In the genome sequence of this NBRC4239 strain, the xlnR gene (DOGAN gene number: AO090012000267, (http://www.nbrc.nite.go.jp/dogan/) and the manR gene disclosed in the Aspergillus oryzae RIB40 strain A search was performed using the BLAST program (Nucleic Acids Res., 25, 3389? 402, 1997) to see if a gene corresponding to (DDBJ / ENA / Genbank accession number: AB70164) exists.

As a result, on the genomic DNA of Aspergillus soya, the Aspergillus soya manR gene shown in SEQ ID NO: 1 ( scaffold No. 11, DDBJ accession number DF093563.1: 511177-588691) and the Aspergillus soya manR shown in SEQ ID NO: 2 It was confirmed that the soya xlnR gene ( scaffold No. 48, DDBJ accession number DF093577.1: 2111489-2114520) was present.

Moreover, based on the exon-intron structure of each transcription factor gene in Aspergillus oryzae, the exon-intron structure of the manR and xlnR genes in Aspergillus soja was predicted. Based on the data, the sequence of amino acids encoded by each gene was estimated using GENETYX software version 11 (manufactured by Genetics Co., Ltd.).

As a result, it was expected that the manR gene of SEQ ID NO: 1 encodes the amino acid sequence shown in SEQ ID NO: 3, and the xlnR gene of SEQ ID NO: 2 encodes the amino acid sequence shown in SEQ ID NO: 4. The identity of Aspergillus soya ManR (SEQ ID NO: 3) and Aspergillus oryzae ManR (AB70164) was 98%, and a difference of 8 amino acids was observed (763/771). Further, the identity of Aspergillus soya XlnR (SEQ ID NO: 4) and Aspergillus oryzae XlnR (AO090012000267) was 97%, and 27 amino acid differences were observed (922/949). From this, it is considered that ManR and XlnR have different sequences in Aspergillus soya and Aspergillus oryzae.

Manufacture of manR - xlnR double compulsory expression strain in Aspergillus soya in soy sauce koji When the genes shown in SEQ ID NO: 1 and SEQ ID NO: 2 are forcibly expressed simultaneously in soy sauce koji, cellulase and hemicellulase of this fungus It was verified whether there was an effect of enhancing production capacity.

・ Medium used for production of forced expression strain As the culture medium for Aspergillus soya, dextrin-peptone medium (polypeptone 1%, dextrin 2%, potassium dihydrogen phosphate 0.5%, sodium nitrate 0.1% , Magnesium sulfate 0.05%, casamino acid 0.1%, pH 6.0).

In addition, the minimal agar medium used for culturing this bacterium includes Czapek-Dox minimal medium (3.0% glucose, 0.05% KCl, 0.2% NaNO ₃ , 0.1% KH ₂ PO ₄ , 0 0.05% MgSO ₄ , 0.001% FeSO ₄ , pH 6.0).

  In addition, when forming conidia of this bacterium, malt agar medium (malt extract 8%, copper sulfate 0.0002%, sodium tetraborate 0.00004%, ferric phosphate 0.00087%, manganese sulfate 0.00095%, sodium molybdate 0.0008%, zinc sulfate 0.0008%, agar 1.5%, pH 6.0).

  When the auxotrophic strain was cultured, uridine having a final concentration of 15 mM was added to a medium using uridine-requiring strain, and a final concentration of 0.01% adenine was added to an adenine-requiring strain.

・ Method of preparing genomic DNA from Aspergillus soja cells of soy sauce koji Aspergillus soya NBRC4239 strain DNA used for Fusion PCR template DNA at the time of vector production is extracted and purified by the following procedure It was. Filamentous fungi or conidia were planted in 40 ml of dextrin-peptone medium in a 150 ml Erlenmeyer flask and cultured at 30 ° C. and 150 rpm for 3 days. After completion of the culture, the bacterial cells were collected by filtration, sandwiched between paper towels to remove moisture, and then rapidly frozen using liquid nitrogen.

  Next, the frozen cells were poured into a mortar that had been cooled by pouring liquid nitrogen, and were carefully crushed using a pestle that had been cooled with liquid nitrogen. From this pulverization, total DNA was extracted using Genomic DNA extraction Kit (Promega), and then the sample was treated with DNase free RNase (Nippon Gene) to decompose the contaminated RNA. Used for.

-Preparation of genomic DNA from conidia of soy sauce koji Aspergillus soya Cell bodies were cultured on Marz agar medium at 30 ° C for 7 days to form conidia. The conidia were collected after being suspended in 5 mL of a sterilized 0.01% Tween solution per plate. After centrifugation of the obtained conidia suspension (500 μL), the precipitate was resuspended in 800 μL of Nuclei Lysis Solution (manufactured by Promega), and this was filled with glass beads having a diameter of 0.5 mm (500 μL). It moved to 2.0 mL with a screw cap, and crushed with a bead shocker (manufactured by Yasui Equipment Co., Ltd.). After 650 μL of the disrupted solution was collected, 300 μL of Protein Precipitation Solution (Promega) was added and mixed, then centrifuged at 12,000 × g for 15 minutes, and 650 μL of the supernatant was collected. 650 μL of phenol / chloroform / isoamyl alcohol (Nippon Gene Co., Ltd.) was added thereto, followed by vigorous mixing with a vortex mixer, followed by centrifugation at 12,000 × g for 10 minutes. 600 μL of the aqueous layer was collected and concentrated by 2-propanol precipitation, and then the precipitate was dissolved in 50 μL of TE buffer to obtain genomic DNA.

-Production of host strain used in this Example 2 and background of its production The manR-xlnR double compulsory expression strain described in Example 2 is an Aspergillus soja KPA-del-S2-5R strain (Δ pyrG , Δ adeA , Δku70 ) (Aspergillus soja NBRC4239 strain derivative ) was used as a host.

Ltd. In addition, Aspergillus sojae KPA-del-S2-5R strain, which was produced Aspergillus sojae KP-del strain (Δ pyrG, Δ ku70) a (NBRC4239 shares derivatives) (Kikkoman Corporation donated by Junko Manaka) as a parent strain It is.

The Aspergillus soja KP-del strain ( ΔpyrG , Δku70 ) is a strain obtained through the following procedure (FIG. 1).

The Aspergillus soja NBRC4239 strain was cultured on a medium containing perchloric acid to obtain a strain in which a natural mutation was introduced into the niaD gene and inactivated (Aspergillus soja ΔniaD strain). An Aspergillus soja ΔpyrG , niaD + strain was prepared by introducing a niaD gene into which no mutation was introduced into the pyrG locus of this ΔniaD strain.

The delta pyrG, the ku70 gene niaD + strains, then disrupted by pyrG gene, performs removal of pyrG gene, delta ku70, was obtained Aspergillus sojae P6-1-12 strain is delta pyrG strain.

Next, this P6-1-12 strain to remove the niaD gene that has been introduced into the pyrG locus delta ku70, delta pyrG, were obtained ND-del strain is delta niaD strain. ND-del strain a mutant niaD gene present in, replacing the niaD gene mutation is not introduced, Aspergillus sojae KP-del strain (delta ku70 is a strain of state niaD gene was restored to wild type, delta pyrG Obtained).

Aspergillus sojae KPA-del-S2-5R strain (Δ pyrG, Δ adeA, Δ ku70) is a strain prepared by the steps shown below (Figure 2).
In the Aspergillus soja KP-del strain, pyrG was introduced into the adeA locus in a state where it could be removed by loop-out. The adeA removal vector was prepared using the Fusion PCR method.

  The reaction was carried out in a total volume of 40 μL using TOYOBO KOD Plus Neo as the PCR enzyme and the genome derived from Aspergillus soja NBRC4239 (20 ng per reaction) as the template. The primer sequences used are as shown in Tables 1-4. The temperature cycle conditions for 1st PCR were 94 ° C. for 2 minutes, followed by 35 cycles of 98 ° C. for 10 seconds, 60 ° C. for 10 seconds, and 68 ° C. for 5 minutes.

  The total amount of the obtained PCR product was subjected to electrophoresis and then purified using a gel extraction kit manufactured by QIAGEN. 20 μL of each of these purified DNA fragments was mixed, and 16 μL of this was used as a template for Fusion PCR. SEQ ID NO: 5 and SEQ ID NO: 12 were used as primer sets for performing Fusion PCR. As the PCR enzyme, KOD Plus neo was used in the same manner as the 1st PCR, and the reaction was performed in a total volume of 320 μL. Fusion PCR temperature cycle conditions were 94 ° C. for 2 minutes, followed by 98 ° C. for 10 seconds and 68 ° C. for 10 minutes for 35 cycles. The amplified adeA disruption vector was concentrated by 2-propanol precipitation and dissolved in 10 μL of TE buffer. This was introduced into the Aspergillus soja KP-del strain using the protoplast-PEG method.

In this transformation, since the vector has a pyrG marker, the resulting strain is a pyrG + strain. However, since adeA is destroyed, the final concentration of the selective medium is 0.01%. A Czapek-Dox minimal medium containing 1.2 M sorbitol to which adenine was added was used. The obtained transformant was planted twice in a Czapek-Dox minimal medium containing adenine and then planted in a Marz agar medium to form conidia. These conidia were suspended in 5 mL of a sterilized 0.01% Tween solution per plate and collected, and genomic DNA was extracted and purified. PCR was performed using this genomic DNA as a template and SEQ ID NO: 5 and SEQ ID NO: 12 as oligonucleotide primers, and it was confirmed whether the vector was introduced into the target region. As a negative control, Aspergillus soja KP-del strain was used.

As a result, 4 strains into which the vector was correctly introduced were obtained. These four strains were all strains showing adenine requirement. The present transformant Aspergillus sojae KA-del named (Δ ku70, Δ adeA, pyrG +) and.

Aspergillus soja KA-del was planted on Marz agar medium and cultured at 30 ° C. for 7 days, and conidia were collected. The conidia were diluted with a sterilized 0.01% Tween solution to 2.5 × 10 ⁵ / mL. 200 μL of this diluted solution was spread with a spreader in a Tuapeck Czapek-Dox minimal medium containing 5-fluoroorotic acid (5-FOA) at a final concentration of 1.5 mg / mL, 15 mM uridine and 0.01% adenine, and 4 ° C. at 30 ° C. For a day. The obtained 5-FOA resistant strain was cultured again on Marz agar medium, and then genomic DNA was extracted from conidia. Using this genomic DNA as a template, by performing PCR using the oligonucleotide primers shown in SEQ ID NO: 13 and SEQ ID NO: 14 (both primers designed outside the deletion region), a loop-out type containing the deletion of the pyrG gene It was confirmed whether recombination had occurred. As a result, it was found that the desired recombinant was obtained in a plurality of strains (FIG. 3A).

  For the S2-5R strain, which is one of the strains, the DNA deletion region of the genome and the surrounding DNA region were analyzed (FIG. 3A). As a result, it was confirmed that recombination occurred as originally designed in this strain. Further, when the auxotrophy of this strain was examined, it was confirmed that it was a double auxotrophic strain of adenine and uridine (FIG. 3B).

The strain Aspergillus sojae KPA-del-S2-5R (Δ ku70 , Δ pyrG, Δ adeA Ltd.) named, ManR - was used as an xlnR double forced expression strain hosts for producing.

In addition, a strain in which the pyrG gene of the Aspergillus soja KP-del strain was restored to the same as the wild type was prepared. Using the genomic DNA of Aspergillus soja NBRC4239 strain as a template and the oligonucleotide primers shown in SEQ ID NOs: 15 and 16, a 6.6 kb DNA fragment containing the full length of the pyrG gene was introduced to prepare a strain in which the pyrG gene was restored. The results of confirming the restoration of the pyrG gene of this strain by PCR are shown in FIG.

In addition, SEQ ID NO: 15 and SEQ ID NO: 16 were used as PCR primers for confirmation. As a result of electrophoresis, it was confirmed that a plurality of host strains in which the pyrG gene was restored could be obtained. The transformant as Aspergillus sojae NBRC4239deruta ku70 strain was used as a negative control in performing confirmation of the effect of manR and xlnR forced expression of.

・Preparation of xlnR forced expression cassette introduction strain
An outline of the vector for forced expression of xlnR is shown in FIG.
Since the target gene to be forcibly expressed this time is a transcriptional regulatory factor, there is a concern that increasing the copy number of the vector will overload the cells and cause adverse effects. Therefore, the original promoter of xlnR was replaced with a stronger promoter of alkaline protease gene so that xlnR was highly expressed only during solid culture.

The xlnR forced expression vector was prepared using the Fusion PCR method. The reaction was performed in a total amount of 40 μL using KYO Plus Neo from TOYOBO as the PCR enzyme and the genome derived from Aspergillus soja NBRC4239 (20 ng per reaction) as the template.

  The primer sequences used are as shown in Tables 7-10. The temperature cycle conditions of the 1st PCR were 94 ° C. for 2 minutes, followed by 35 cycles of 98 ° C. for 10 seconds, 60 ° C. for 10 seconds, and 68 ° C. for 5 minutes.

  The total amount of the obtained PCR product was subjected to electrophoresis and then purified using a gel extraction kit manufactured by QIAGEN. 20 μL of each purified DNA fragment was mixed, and 16 μL of the purified DNA fragment was used as a template for Fusion PCR. SEQ ID NO: 17 and SEQ ID NO: 24 were used as primer sets for performing Fusion PCR. As the PCR enzyme, KOD Plus neo was used in the same manner as 1st PCR, and the reaction was carried out in a total volume of 40 μL. Fusion PCR temperature cycle conditions were 94 ° C. for 2 minutes, followed by 98 ° C. for 10 seconds and 68 ° C. for 10 minutes for 35 cycles.

  When the Fusion PCR product was confirmed by agarose gel electrophoresis, only a small amount of the vector for forced expression was obtained. Therefore, the amplified Fusion PCR product was purified by gel extraction and then used as a template, and nested PCR was performed using the primers shown in Table 11.

  As a result, the target forced expression vector was successfully amplified (reaction conditions were the same as those of Fusion PCR except that the reaction volume was increased to 320 μL).

The amplified xlnR forced expression vector was concentrated by 2-propanol precipitation and dissolved in 10 μL of TE buffer. This was introduced into Aspergillus soja KPA-del-S2-5R strain using protoplast-PEG method. In this transformation, since the vector has an adeA marker, the resulting strain is an adeA + strain, but since pyrG remains destroyed, the final concentration of uridine in the selective medium is 15 mM. Czapek-Dox minimal medium containing 1.2M sorbitol supplemented with. Using this genomic DNA as a template, PCR was performed using the oligonucleotide primers shown in SEQ ID NO: 25 and SEQ ID NO: 26, and it was confirmed whether the vector was introduced into the target region. As a negative control, the host strain Aspergillus soja KPA-del-S2-5R was used. The confirmation result of the transformant by PCR is shown in FIG. The obtained transformant was in a state in which the vector was correctly introduced and the nucleus was purified. The transformant was Aspergillus sojae xlnR -OEΔ pyrG strain.

· XlnR introduction of ManR forced expression cassette to forced expression strain Subsequently, the xlnR -OEderuta pyrG strain as a host, ManR - were prepared xlnR double forced expression strain. The structure of the manR forced expression vector is shown in FIG. Since manR a transcription factor gene as with xlnR also, in the case of performing the forced expression with increased copy number, that there is a negative effect on the cells it is concerned. Therefore, gene forced expression was performed by replacing the original promoter of the manR gene with a promoter that is a translation elongation factor promoter.

The manR forced expression vector was prepared using the Fusion PCR method in the same manner as the xlnR forced expression vector. The PCR primers used for the preparation of the manR forced expression vector are as shown in Tables 12 to 15, and the other reaction conditions are the same as the construction of the xlnR forced expression vector.

  The obtained PCR product was purified and mixed in an equal volume of 20 μL, and 16 μL of that was used as a template for Fusion PCR. SEQ ID NO: 27 and SEQ ID NO: 34 were used as primer sets when performing Fusion PCR. As an enzyme for PCR, TOYOBO KOD Plus neo was used, and the reaction was performed in a total volume of 320 μL. Fusion PCR temperature cycle conditions were as follows: heating at 94 ° C. for 2 minutes, followed by 35 cycles of 98 ° C. for 10 seconds and 68 ° C. for 10 minutes.

When the obtained Fusion PCR product was confirmed by agarose gel electrophoresis, the desired PCR product was obtained satisfactorily. Accordingly, after which was concentrated by alcohol precipitation, xlnR -OEΔ pyrG strain was introduced (Δ ku70, Δ pyrG, adeA + strain) to. Since the pyrG marker is mounted on the manR forced expression vector, the manR - xlnR double forced expression strain is a strain having no auxotrophy. The obtained transformants as well as Aspergillus sojae xlnR -OEΔ pyrG strain was confirmed by PCR method. SEQ ID NO: 27 and SEQ ID NO: 34 were used as primers. Further, for the obtained manR - xlnR double forced expression strain, SEQ ID NO: 25 and SEQ ID NO: 26 described in Table 11 were used, and it was also confirmed by PCR whether the xlnR forced expression cassette was correctly maintained. .

The result of confirmation of the transformant is shown in FIG. As a negative control, Aspergillus soja NBRC 4239 strain was used. A plurality of transformants obtained, is introduced forced expression vector cassette and xlnR forced expression vector cassette manR is correctly and could nuclei also in a state of being purified was confirmed. This transformant was designated as Aspergillus soja manR - xlnR- OE strain.

・Production of Aspergillus soya with forced expression of manR alone
For ManR alone forced expression strain, ManR - xlnR genomes -OE strain as a template, the cassette ManR forced expression was amplified by PCR, Aspergillus sojae KP-del (Δ ku70, Δ pyrG) to introduce into the strain Produced. SEQ ID NO: 27 and SEQ ID NO: 34 were used as oligonucleotide primers for PCR, and KOD Plus Neo from TOYOBO was used as an enzyme for PCR.

The confirmation of the transformant is shown in FIG. As a negative control, Aspergillus soja KP-del strain was used. It was possible to obtain a plurality of manR single forced expression strains that were nuclear-purified and had no auxotrophy . This manR single forced expression strain was designated as Aspergillus soja manR- OE strain.

-Production of xlnR single forced expression strain without auxotrophy
For xlnR -OEΔ pyrG strains with auxotrophic, this strain ManR - seems it is not appropriate to be used as a comparative control of xlnR -OE strains. So to produce the xlnR -OEderuta pyrG strain strain is restored the pyrG gene. PCR was performed using Aspergillus soja NBRC4239 strain genomic DNA as a template and the oligonucleotide primers (SEQ ID NO: 15 and SEQ ID NO: 16) shown in Table 6 to amplify a 6.6 kb DNA fragment containing the full length of the pyrG gene. . The temperature cycle conditions for PCR were heating at 94 ° C. for 2 minutes, followed by 35 cycles of 98 ° C. for 10 seconds and 68 ° C. for 9 minutes. The resulting DNA fragments After concentration by 2-propanol precipitation were introduced into xlnR -OEΔ pyrG strain to obtain a transformant by selection on minimal medium containing 1.2M sorbitol. This transformant was confirmed by xlnR -OEΔ pyrG strain produced at the same way.

The confirmation of the transformant is shown in FIG. The negative control was used xlnR -OEΔ pyrG. A plurality of xlnR- only forced expression strains that were nuclear-purified and no auxotrophy were obtained. This xlnR single forced expression strain was designated as Aspergillus soja xlnR- OE strain.

・ Measurement of various glycolytic enzyme activities of strains forcibly expressing transcriptional regulatory factor genes In the transformant prepared in Example 2, the target transcriptional regulatory factors were correctly expressed and the production of various glycolytic enzymes It was verified whether an increase occurred.

(1) Preparation of soy sauce cake and preparation of extracellular enzyme extract Each transformant was inoculated on Marz agar medium and cultured at 30 ° C. for 7 days to form conidia. The conidia are collected using a sterilized 0.01% Tween solution, passed through a sterilized 70 μm mesh cell strainer (BD Falcon), and the mixed mycelia are removed. Then, using a hemocytometer The number of conidia in each suspension was counted. Based on this measured value, the suspension was diluted with a 0.01% Tween solution so that the number of conidia of each suspension was 5.0 × 10 ⁷ / mL. 7 g of raw material (defatted soybean: wheat: water = 1: 1: 1.3) was placed in a 150 mL Erlenmeyer flask and sterilized by autoclaving. This was inoculated with 200 μL of the diluted conidia and cultured at 30 ° C. for 40 hours. 20 mL of 20 mM sodium phosphate buffer pH 7.0 was added to a 150 mL Erlenmeyer flask after completion of the culture, and a rubber stopper was added, followed by vigorous stirring 100 times. This was allowed to stand at 4 ° C. overnight, and ADVANTEC no. The mixture was filtered through 5C filter paper, and the filtrate was used for enzyme activity measurement.

(2) Various hydrolase activity measurement procedures β-mannanase activity measurement In 150 μL of a substrate solution in which azo-carob galactomannan (megazyme) was dissolved to 50% sodium acetate buffer (pH 4.5), 75 μL A 50 mM sodium acetate buffer (pH 4.5) and 75 μL of a crude enzyme solution appropriately diluted with a 50 mM sodium acetate buffer were added, and the mixture was incubated at 40 ° C. for 30 minutes. 800 μL of 100% ethanol was added thereto, allowed to stand for 10 minutes, centrifuged at 1,500 × g for 15 minutes, and then the mannanase activity was measured by measuring the OD590 nm of the supernatant. In addition, as a blank, what added 100% ethanol for reaction stop before the enzyme solution was used. Moreover, the minus the OD590nm of the blank from OD590nm of each specimen and DerutaOD590nm, and 1 times the average value of DerutaOD590nm in control (NBRC4239Δ ku70), was determined relative activity of each sample.

Measurement of xylanase activity 50 μL of a crude enzyme solution diluted with 50 mM sodium acetate buffer (pH 4.5) in 250 μL of a substrate solution in which RBB xylan was dissolved at 0.25% in 50 mM sodium acetate buffer (pH 4.5) Was added and incubated at 40 ° C. for 20 minutes. To this, 1,000 μL of 100% ethanol was added, allowed to stand at room temperature for 10 minutes, and then centrifuged at 15,000 × g for 10 minutes. The xylanase activity was calculated | required by measuring OD595nm of the supernatant liquid after this centrifugation. In addition, as a blank, what added 100% ethanol for reaction stop before the enzyme solution was used. Moreover, the minus the OD595nm of the blank from the OD595 nm of each sample and DerutaOD595nm, and 1 times the DerutaOD595nm average value in the control (NBRC4239Δ ku70), was determined relative activity of each sample.

Endoglucanase activity measurement (CMCase activity)
150 μL of a crude enzyme solution diluted 5-fold with a buffer solution is added to 150 μL of a substrate solution in which azo-carboxymethylcellulose (manufactured by Megazyme) is dissolved in 50 mM sodium acetate buffer (pH 4.5) to 2.0%. And incubated at 40 ° C. for 20 minutes. After adding 800 μL of a reaction stop solution (mixture of 2% sodium acetate-0.2% zinc acetate solution pH 5.0 and 99.5% ethanol 1: 4) to the reaction solution, 10 × 1,500 × g Centrifugation was performed for minutes. The endoglucanase activity was measured by measuring the OD590nm of this supernatant. In addition, as a blank, what added the reaction stop solution before the enzyme solution was used. Moreover, the minus the OD590nm of the blank from OD590nm of each specimen and DerutaOD590nm, and 1 times the DerutaOD590nm average value in the control (NBRC4239Δ ku70), was determined relative activity of each sample.

Cellobiohydrolase activity measurement (Avicelase)
Add 300 μL of the crude enzyme solution stock solution to 300 μL of the substrate solution in which azo-Avicel (manufactured by Megazyme) is uniformly dispersed so as to be 6.0% in 50 mM sodium acetate buffer (pH 4.5), and bring it to 40 ° C. And incubated for 24 hours at 180 rpm with shaking. The reaction solution was treated at 95 ° C. for 15 minutes to stop the reaction, and then centrifuged at 1,500 × g for 10 minutes. The cellobiohydrolase activity was determined by measuring the OD590 nm of the supernatant after this centrifugation. In addition, as a blank, what was processed for 15 minutes at 95 degreeC immediately after adding an enzyme solution was used. Moreover, the minus the OD590nm of the blank from OD590nm of each specimen and DerutaOD590nm, and 1 times the DerutaOD590nm average value in the control (NBRC4239Δ ku70), was determined relative activity of each sample.

p - nitrophenyl (p NP) activity assay using a derivative substrate (alpha-galactosidase, and β- xylosidase)
An extracellular enzyme solution appropriately diluted with a buffer solution was added to 400 μL of a synthetic substrate solution ( p -nitrophenyl-derivative of each carbohydrate) dissolved in a 50 mM sodium acetate buffer solution (pH 4.5) so as to have a concentration of 1.25 mM. 100 μL was added and incubated at 40 ° C. for 15 minutes. After stopping the reaction by adding 500 μL of 0.5 M sodium carbonate solution, the absorbance at 405 nm (OD 405 nm), which is the absorption maximum of p -nitrophenol released by the hydrolysis reaction, was measured. Degradative enzyme activity was measured. Moreover, after adding 0.5 M sodium carbonate to a 1.25 mM synthetic substrate solution first, the series which added the diluted enzyme solution was made into the blank. The value obtained by subtracting the OD405 of the blank from OD405nm value of series an enzymatic reaction was carried out as DerutaOD405nm, as one times the average value of DerutaOD405nm in control (NBRC4239Δ ku70), was determined relative activity of each sample.

(3) Measurement result of extracellular enzyme activity The measurement results of β-mannosidase and α-galactosidase activity of each specimen are shown in FIG. In ManR -OE strain, beta-mannanase and α- galactosidase activity as compared to the control strain (NBRC4239Δ ku70), it was elevated significantly. This was also the result of showing that ManR functions as a positive regulator of the mannan hydrolase group in the soy sauce koji Aspergillus soja. This is a phenomenon similar to that previously observed in Aspergillus oryzae (Fungal Genet. Biol .; 49, 987-995, 2012). In the manR - xlnR- OE strain, β-mannanase tended to increase remarkably and α-galactosidase tended to increase.

  The activity measurement of xylanase and β-xylosidase, which are enzymes controlled by XlnR, is shown in FIG.

In the xlnR- OE strain, an increase in xylanase and β-xylosidase activities was confirmed. XlnR also functions as a positive transcriptional regulator of the xylan hydrolase group in Aspergillus sojae, similar to Aspergillus oryzae XlnR (Fungal Genet. Biol .; 35, 157-169, 2002). I understood it. In addition, xylanase activity tended to slightly increase in the manR- only forced expression strain, and the manR - xlnR- OE strain showed higher xylanase activity than the xlnR- OE strain. This suggests that some xylanase genes are controlled by ManR in Aspergillus sojae. Regarding β-xylosidase activity, no increase in activity was observed in the manR single forced expression strain.

However, the manR - xlnR- OE strain was confirmed to have higher activity than the xlnR- only forced expression strain. ManR alone cannot control the production of β-xylosidase, but there is a possibility of positively controlling β-xylosidase in the presence of XlnR. In this example, in manR - xlnR- OE strains, significant increases in β-mannanase activity and xylanase activity were confirmed, and therefore it was determined that manR and xlnR were correctly expressed in this transformant. .

In the case of analysis in the past Aspergillus oryzae, both ManR and XlnR are known to control cellulase gene expression (Biosci. Biotechnol. Biochem .; 77, 426-429, 2013; FEBS Lett. 528). 279-282, 2002). Therefore, it is expected that Aspergillus sojae in which manR and xlnR are expressed by double forced expression has a high cellulase production ability. The cellulase productivity of manR - xlnR- OE was evaluated (FIG. 13).

Regarding endoglucanase activity, the manR - xlnR- OE strain showed the highest activity, and had an activity five times or more that of the control strain. In addition, cellobiohydrolase activity was weaker than endoglucanase because Avicel as a substrate is crystalline cellulose, and it is difficult to decompose, but it is the highest in the manR-xlnR-OE strain. Activity was seen.

As described above, it was confirmed that the manR - xlnR double forced expression in Aspergillus soja significantly increased the amount of cellulase produced by this bacterium. This shows that this double forced expression strain is useful for the degradation of plant-derived polysaccharides. Further, in this example, Aspergillus soya was used as the host of the forced expression strain, but considering the data of this example, the functions of the two transcriptional control factors, ManR and XlnR, are aspergillus aspergillus and Aspergillus oryzae. It is thought that it is preserved among Aspergillus oryzae.

From these facts, it is expected that these two strains are highly conserved with respect to cellulase and hemicellulase production control systems. Therefore, even if Aspergillus oryzae is used as a host for forcedly expressing manR and xlnR , it is considered that the same effect as that obtained when Aspergillus sojae is used as a host is obtained.

・Short- term decomposition test of soy sauce raw material using manR - xlnR double expression strain As shown in Example 2, manR - xlnR double forced expression in Aspergillus soja significantly increased cellulase / hemicerase production ability of this bacterium It has been confirmed that Therefore, a short-term decomposition test of soy sauce raw material using this bacterium was conducted to examine whether this strain is effective in promoting the decomposition of polysaccharide components derived from the raw material in soy sauce production.

(1) Effect of manR - xlnR double expression on soy sauce raw material short-term decomposition test for moromi viscosity After mixing defatted soybeans, wheat and water in a ratio of 1: 1: 1.3, weigh 15 g and add 150 mL The flask was placed in an Erlenmeyer flask, capped and sterilized by autoclave. This, 2.7 x 10 ⁷ cells of ManR - xlnR -OE strain, ManR -OE strain, xlnR -OE strains or NBRC4239 Δ ku70 strain conidia of (negative control) were inoculated, respectively, well mixed (Incorporating) . This was cultured at 30 ° C., and after 46 hours had passed from the filling, it was fermented. Each experiment was repeated three times.

  After thoroughly unraveling the sample with a sterilized shell, add 15 mL of 28% saline solution sterilized by autoclaving to mix well, and then use wrap film and rubber bands for product packaging. Covered. This was decomposed for 36 hours while shaking at 30 ° C. and 180 rpm. After the completion of the decomposition reaction, the sample was transferred to a 25 mL polypropylene tube, and the moromi viscosity was measured with a rotational viscometer (TOKIMEC Viscometer BLII).

FIG. 14A shows the state when the soy sauce cake produced using each specimen is extracted. All specimens were in a state where it was confirmed that the mycelium of Aspergillus had grown to the surface, and no significant growth inhibition occurred under the conditions of this test. Moreover, the conidia formation of soy sauce koji molds tended to be delayed in each gene expression strain, and the tendency was remarkable in the manR- OE strain and the manR - xlnR- OE strain. Moreover, the state of the moromi of each specimen at the end of the short-term decomposition test is shown in FIG. 14B.

In each of the transcriptional factor forced expression strains, the moromi juice portion increased compared to the control, and the viscosity of the moromi tended to decrease. The tendency of manR - xlnR- OE strain was also most notable in moromi.

  The results of measuring the viscosity of each sample for each taste are shown in FIG.

In the manR- OE strain and the xlnR- OE strain, it was confirmed that the viscosity was slightly reduced when compared with the control and the average of triplicate. However, in these two series, a sample in which the effect of forced expression is remarkable and a sample in which the effect is hardly seen are mixed. On the other hand, in the manR - xlnR- OE strain, the viscosity of moromi decreased significantly compared to the control, and the difference between the repeated experiments was tended to be smaller than that of the single forced expression strain.

From the above, when the manR - xlnR- OE strain is used, it is considered that the decomposition of the polysaccharide component derived from the soy sauce raw material is stably promoted and the viscosity of the moromi can be reduced.

(2) Effect of manR - xlnR double expression soy sauce raw material short-term decomposition test on the amount of juice collected by natural dripping After mixing defatted soybeans, wheat and water in a ratio of 1: 1: 1.3, weigh 15 g. Into a 150 mL Erlenmeyer flask, put a cotton plug and sterilized by autoclave. This, 2.7 x 10 ⁷ cells of ManR - xlnR -OE strain, ManR -OE strain, xlnR -OE strains or NBRC4239 Δ ku70 strain conidia of (negative control) were inoculated, respectively, well mixed (Incorporating) . This was cultured at 30 ° C., and after 46 hours had passed from the filling, it was fermented. Each experiment was repeated three times.

  After thoroughly unraveling the sample with a sterilized case, add 15 mL of 28% saline solution sterilized by autoclaving and mix well to make the taste, then use food packaging wrap film and rubber bands. Covered. This was decomposed for 48 hours while shaking at 30 ° C. and 180 rpm. This moromi was used in a Whatman No. 110 mm diameter set in a funnel. About 24 g was collected on 1 filter paper, and the amount was measured with a balance. And the liquid juice obtained by filtration is recovered with a 15 mL polypropylene tube whose weight has been measured in advance, and the total weight is measured over time, so that the liquid juice (raw material decomposition liquid) recovered from the moromi is recovered. ) Was measured. And the amount of the soup obtained from 1g of moromi was calculated | required by dividing the weight of this collect | recovered soup by the moromi weight (g) applied on the filter paper.

FIG. 16A shows the results of measuring the change over time in the amount of juice collected by natural drooling in the raw material short-term decomposition test. In addition, this graph is the figure which plotted the average of the liquid volume measurement value of 3 series, respectively. In the manR- OE strain and the xlnR- OE strain, the amount of recovered juice was larger than that in the control strain. In manR - xlnR- OE, more sap was collected than in the manR- OE strain and xlnR- OE strain.

From this, it was confirmed that the double forced expression of manR and xlnR has an effect on the amount of sap collected by natural dripping in the short-term decomposition of the soy sauce raw material. In addition, FIG. 16B shows the weight of the recovered liquid after 4 hours from the start of filtration, in which the amount of recovered liquid juice did not increase. According to this result, in the manR- OE strain and the xlnR- OE strain, both of the specimens in which the effect of forced expression of the transcriptional regulatory factor is markedly observed and the specimen that is not so effective are observed, as in the case of the moromi viscosity. -In the xlnR- OE strain, the weight of the recovered solution was stably increased.

The data of this example were obtained by directly examining the decomposition of defatted soybean and wheat, which are the raw materials of soy sauce, in a situation where a high concentration of salt exists, and this confirmed that the effectiveness was confirmed. It is considered that there is a high possibility of being effective in the actual soy sauce manufacturing process. From this data, it is considered that the manR - xlnR - OE strain is also useful when a plant-derived material is decomposed in a short period of time in the presence of salt to produce a soy sauce-like seasoning liquid.

-Verification of the effect of manR - xlnR double forced expression in Aspergillus soja NBRC4241 strain line The data shown in Examples 2 to 3 are all data obtained with derivatives of Aspergillus soja NBRC4239 strain. In the case of Aspergillus genus fungus, even if it is the same genus, there may be no effect when gene forced expression is carried out in different strains.
Therefore, it was verified whether manR - xlnR double forced expression was effective even when a strain of Aspergillus soja NBRC4241 strain, which is a different series from NBRC4239 strain, was used as a host.

Soy sauce koji mold Aspergillus sojae DKuAP-1 strain; a (Δ Ku70, Δ adeA, Δ pyrG strain mutants to the parent strain Aspergillus sojae NBRC4241 strain) as the host, a xlnR alone forced expression strain XROE-N2 strain and ManR - The MXOE -N2 strain, which is a xlnR double forced expression strain, was prepared. Further, ManR alone for forced expression a strain MXOE-N2 strain, soy sauce koji mold Aspergillus sojae DKuP-1 strain (Δ Ku70, Δ pyrG strain; Aspergillus sojae DkuAP-1 strain adenine auxotrophic no strain) host And made.

  These transformants were prepared in the same procedure as the preparation of various forced expression strains in the Aspergillus soja NBRC4239 strain described in Example 2. The transformants used in Example 4 were confirmed to be in a state where the vector was correctly introduced and the nuclei were purified (FIG. 17).

Further, as a control, ADEA and pyrG Aspergillus sojae DKuAP-1 strain is a host strain is a strain that has not been destroyed, using Aspergillus sojae DKu strain (Δ ku70).

-Soy sauce raw material short-term degradation test using MXOE-N2 strain A short-term degradation test was conducted to evaluate whether the transformant was effective in degrading soy sauce raw materials. After mixing defatted soybeans, wheat and water in a ratio of 1: 1: 1.3, 15 g was weighed, placed in a 150 mL Erlenmeyer flask, capped and sterilized by autoclave. This was inoculated with 2.7 × 10 ⁷ conidia of XROE-N2 strain, MROE-N2 strain, MXOE-N2 strain or Aspergillus soja Dku strain (control), and mixed well (incorporated). This was cultured at 30 ° C., and after 43 hours from filling, it was fermented. Each experiment was repeated three times.

  After thoroughly unraveling the sample with a sterilized case, add 15 mL of 28% saline solution sterilized by autoclaving and mix well to make the taste, then use food packaging wrap film and rubber bands. Covered. This was decomposed for 64 hours while shaking at 30 ° C. and 180 rpm.

-Moromi viscosity measurement After completion | finish of decomposition reaction, the sample was moved to a 25 mL volume polypropylene tube, and the viscosity was measured with the rotational viscometer (TOKIMEC Viscometer BLII).

・ Measurement of the amount of sap by natural dripping After the short-term decomposition reaction, the moromi taste was set to 110 mm diameter Whatman No. About 25 g was collected on 1 filter paper, and the amount was measured with a balance. The sap obtained by filtration is recovered by a 15 mL polypropylene tube whose weight has been measured in advance, and the total weight of the sap is appropriately measured over time. The amount of was measured. By dividing the weight of the recovered sap by the moromi weight (g) applied on the filter paper, the amount of sap obtained from 1 g of moromi was obtained.

-Results of raw material short-term decomposition test FIG. 18A shows the results of measuring the moromi viscosity. In the MXOE -N2 strain, which is a manR- xlnR double compulsory expression strain, the moromi viscosity was significantly reduced compared to other specimens.

  FIG. 18B shows the change over time in the amount of juice obtained by filtering the moromi in the short-term decomposition test.

  In addition, a graph plots the average value of the decomposition | disassembly test done in triplicate. When the moromi when using MXOE-N2 strain was filtered, the largest amount of sap was recovered.

From the above data, the effect of manR - xlnR double forced expression is not limited to when the Aspergillus soja NBRC4239 strain is used as a host, but when other Aspergillus soya is used as a host. Is also considered to appear.

・ Soy sauce small-scale brewing test using MXOE-N2 strain
ManR - xlnR using MXOE-N2 strain is a double forced expression strain soy sauce upon Fermentation was confirmed whether increasingly lower and natural sag by recovery of juice of moromi viscosity is observed.

-Shoemaking 55 g of soy sauce raw material in which defatted soybean, wheat and water were mixed at a ratio of 1: 1: 1.3 was placed in a Fernbach flask, sterilized by an autoclave and cooled to room temperature. Here, 2.5 mL of conidia of XROE-N2, MROE-N2, MXOE-N2, and control strain (Dku strain) diluted to 4.0 × 10 ⁷ / mL were inoculated and mixed well ( Filling). This was incubated at 30 ° C. for 18 hours and then loosened after one care, then incubated at 30 ° C. for another 6 hours and then double-cured and loosened again. After the second care, the temperature was lowered to 25 ° C., followed by culturing for 19 hours.

-Preparation, lactic acid fermentation, yeast fermentation 40 g of the fermented rice sample was transferred to a sterilized 100 mL plastic bottle, and 35 mL of 30% saline (w / v) was added thereto and mixed to make moromi. To this was added purely cultured soy sauce lactic acid bacteria Tetragenococcus halofilus and soy sauce yeast Digosaccharomyces luxi and fermented at 15 to 30 ° C. for 90 days.

  During the fermentation test, the series in which the moromi viscosity decreased the earliest was the MXOE-N2 strain. The state of moromi which fermented for 90 days was shown in FIG.

  Each gene forced expression strain tended to have a larger portion of the juice than the control, and the MXOE-N2 strain was most prominent.

-Measurement of moromi viscosity The moromi of fermentation for 90 days was transferred to a 25 mL polypropylene tube and kept at 25 ° C for 1 hour. The viscosity of the moromi that had been kept warm was measured with a rotational viscometer (TOKIMEC Viscometer BLII).

  The measurement results of the moromi viscosity are shown in FIG.

  In addition, this table | surface shows the average measured value and standard deviation of the preparation sample performed in triplicate. From the viewpoint of the average viscosity, each gene forced expression strain tended to have a lower moromi viscosity than the control. In particular, in the XROE-N2 and MXOE-N2 strains, the moromi viscosity tended to decrease remarkably, and the moromi viscosity was the lowest in the MXOE-N2 strain, which is a double compulsory expression strain. .

On the other hand, in the series of the MROE-N2 strain, there was a large variation in the moromi viscosity between the series. From the above, it has been found that the double forced expression of manR and xlnR is effective in reducing the moromi viscosity even under actual soy sauce charging conditions.

・ Measurement of juice volume by natural dripping 110mm diameter Whatman No. 110 was set in a funnel with the soy sauce moromi that had been fermented. About 24 g was collected on 1 filter paper, and the amount was measured with a balance. The sap obtained by filtration is collected with a 15 mL polypropylene tube whose weight has been measured in advance, and the total weight is measured over time, so that the sap is recovered from the moromi by natural dripping (raw material decomposition solution). The amount of was measured. By dividing the weight of the recovered sap by the moromi weight (g) applied on the filter paper, the amount of sap obtained from 1 g of moromi was obtained.

  FIG. 21A shows the results of measuring the change over time in the amount of liquid juice collected from the soy sauce moromi in natural soup, and the collection of each specimen when the change in the liquid amount is no longer seen after 6 hours from the start of filtration. The amount of juice is shown in FIG. 21B. In this test, natural drool is measured for each sample of the fermentation test conducted in triplicate, and the data is tabulated. As a result, in the XROE-N2 strain and the MX-OE strain, the amount of juice collected was significantly increased compared to the control. In addition, many XROE-N2 strains collect liquid at the same level as MXOE-N2 strains, but if there are few strains, there is a tendency for the variation in each charged lot to increase to the same level as the control strain. It was. On the other hand, in the MXOE-N2 strain, there was little difference for each charged lot, and the amount of recovered liquid juice was stably increased.

Thus, when manR and xlnR are expressed forcibly in Aspergillus soya, even when lactic acid fermentation or yeast fermentation is performed, the moromi viscosity is stably reduced and the amount of recovered juice is increased due to natural dripping. It was confirmed.

The method for degrading a polysaccharide using the manR - xlnR double forced expression strain of the present invention significantly accelerates the degradation of the polysaccharide as compared with the case where a single forced expression strain of manR and xlnR is used. In particular, when such a polysaccharide decomposition method was applied to a soy sauce production method, it was confirmed that the moromi viscosity decreased and the amount of juice recovered by natural dripping increased. The results shown in the above examples show that the polysaccharide decomposition method according to the present invention and the soy sauce production method are superior to the methods using conventional strains, and the industrial applicability is high. It shows that.

Claims (4)

A method for degrading polysaccharides comprising:
Using a transformant in which the following genes (a) and (b) are forcibly expressed in Aspergillus sojae or Aspergillus oryzae , the gene (a) is a promoter of a translation elongation factor. And the gene (b) is under the control of an alkaline protease promoter :
(a) any of the following genes (a-1) to (a-5):
(a-1) a gene comprising the nucleotide sequence set forth in SEQ ID NO: 1,
(a-2) a gene encoding the amino acid sequence set forth in SEQ ID NO: 3,
(a-3) a gene encoding a carbohydrate hydrolase transcriptional regulatory factor, comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence set forth in SEQ ID NO: 1;
(a-4) a gene encoding a carbohydrate hydrolase transcriptional regulatory factor, comprising an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 3, or
(a-5) a gene encoding a carbohydrate hydrolase transcriptional regulatory factor consisting of an amino acid sequence in which one or several amino acids of SEQ ID NO: 3 are deleted, substituted and / or added; and
(b) any of the following genes (b-1) to (b-5):
(b-1) a gene comprising the base sequence described in SEQ ID NO: 2,
(b-2) a gene encoding the amino acid sequence set forth in SEQ ID NO: 4,
(b-3) a gene encoding a carbohydrate hydrolase transcriptional regulatory factor, comprising a nucleotide sequence having 90% or more identity with the gene consisting of the nucleotide sequence of SEQ ID NO: 2;
(b-4) a gene encoding a carbohydrate hydrolase transcriptional regulatory factor comprising an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 4, or
(b-5) A gene encoding a carbohydrate hydrolase transcriptional regulatory factor consisting of an amino acid sequence in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 4 have been deleted, substituted and / or added. The method according to claim 1 or 2, wherein the host is Aspergillus sojae . Look including the following genes (a) and (b), under the rule of the gene (a) is a translation elongation factor promoter, and the gene (b) is under the control of the promoter of the alkaline protease, the set Replacement vector.
(a) any of the following genes (a-1) to (a-5):
(a-1) a gene comprising the nucleotide sequence set forth in SEQ ID NO: 1,
(a-2) a gene encoding the amino acid sequence set forth in SEQ ID NO: 3,
(a-3) a gene encoding a carbohydrate hydrolase transcriptional regulatory factor, comprising a nucleotide sequence having 90% or more identity with the gene consisting of the nucleotide sequence set forth in SEQ ID NO: 1;
(a-4) a gene encoding a carbohydrate hydrolase transcriptional regulatory factor, comprising an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 3, or
(a-5) a gene encoding a carbohydrate hydrolase transcriptional regulatory factor consisting of an amino acid sequence in which one or several amino acids of SEQ ID NO: 3 are deleted, substituted and / or added; and
(b) any of the following genes (b-1) to (b-5):
(b-1) a gene comprising the base sequence described in SEQ ID NO: 2,
(b-2) a gene encoding the amino acid sequence set forth in SEQ ID NO: 4,
(b-3) a gene encoding a carbohydrate hydrolase transcriptional regulatory factor, comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence set forth in SEQ ID NO: 2;
(b-4) a gene encoding a carbohydrate hydrolase transcriptional regulatory factor comprising an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 4, or
(b-5) A gene encoding a carbohydrate hydrolase transcriptional regulatory factor consisting of an amino acid sequence in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 4 have been deleted, substituted and / or added. A transformant comprising the recombinant vector according to claim 3 .