WO2013021811A1

WO2013021811A1 - Bacterium having overlapped genomic region

Info

Publication number: WO2013021811A1
Application number: PCT/JP2012/068664
Authority: WO
Inventors: 高橋　理; 敦史佐藤
Original assignee: キッコーマン株式会社
Priority date: 2011-08-05
Filing date: 2012-07-24
Publication date: 2013-02-14
Also published as: JP2013034416A

Abstract

The purpose of the present invention is to enable the production of an yeast fungus having a novel trait, which cannot be obtained so far, in a steady and systematic manner on the basis of a technique for overlapping an arbitrary genomic region on a chromosome of a fungus belonging to the genus Aspergillus. The present invention relates to a transformant of a fungus belonging to the genus Aspergillus, which can produce a wide variety of groups of enzymes necessary for the production of soy source, e.g., proteases, at high levels, said transformant being characterized by having overlapped genomic regions in tandem on the same chromosome and also having a transformed marker gene integrated into a region sandwiched by the overlapped genomic regions.

Description

Bacteria with overlapping genomic regions

The present invention relates to a transformed bacterium belonging to the genus Aspergillus, which has an overlapping genomic region on the same chromosome.

Aspergillus sojae (Aspergillus sojae 工業) and Aspergillus oryzae (Aspergillus 、 oryzae) and other koji molds are widely used industrially for brewing traditional foods such as soy sauce, sake, miso, and enzyme production, etc. In addition, with the recent progress in the determination of the entire genome sequence of Aspergillus oryzae and the comprehensive gene expression analysis using microarrays, the productivity of enzymes, etc. can be improved by genetic engineering modifications, especially at the chromosome level. It is a filamentous fungus that is expected to have an effect of improving the growth rate.

Furthermore, enzymes produced by Aspergillus are used in various industries. For example, in the production of soy sauce, a traditional Japanese food, various enzymes produced by Aspergillus are used. In soy sauce production, koji molds are grown on soybeans and wheat as raw materials to produce various enzymes. Here, the various enzymes produced by Aspergillus decompose proteins, sugars, lipids, etc. of soybeans and wheat, and promote lactic acid fermentation and yeast fermentation in the next process. In this process, when the koji mold produces a large amount of the raw material degrading enzyme, the raw material utilization rate and the squeezability can be increased and the productivity can be greatly improved. In addition, since the substrate for lactic acid fermentation and yeast fermentation is sufficiently supplied, fermentation is performed properly and the quality of soy sauce is greatly improved.

Aspergillus nidulans, niger, fumigatus, awamori and other Aspergillus species have a mononuclear generation, while Aspergillus sojae and Aspergillus oryzae gonococci, including conidia, are always multinucleated in their life cycle So far, no sexual generation has been confirmed, and the mechanism of nuclear distribution from parent cells to daughter cells has not been elucidated. Therefore, new mutants cannot be created by means such as crossing between strains or RIP (repeatrepinduced mutation), genetic research is difficult, and as described above, industrially extremely useful bacteria Nevertheless, genetic analysis was delayed.

From these facts, breeding useful koji molds with high productivity of various enzymes is extremely important in industry, and breeding for this purpose has been vigorously carried out so far. Such gonococcal breeding methods can be broadly classified into mutation methods and gene recombination methods.

Various mutation treatments such as X-rays, ultraviolet rays, and heavy ion beams are used in the mutation method, and screening using the useful properties as an index makes it possible to obtain various strains with excellent enzyme activity and brewing characteristics. Has been created. In recent years, genome information has been used to analyze strains with these useful traits, and the knowledge gained has revealed that chromosome duplication is important for conferring favorable properties on Neisseria gonorrhoeae. As a result of the treatment, Neisseria gonorrhoeae having a large genomic region overlap of 900 kb or more has been obtained (Patent Document 1). Moreover, according to recent research, when yeast obtained by gamma ray irradiation was analyzed, it was reported that repeated sequences exist frequently in the boundary region of the overlapping region of chromosomes (Non-patent Document 1).

However, the mechanism of such chromosomal duplication has not been elucidated. Therefore, in the conventional method using mutation treatment, when screening is performed using enzyme activity or the like as an index, a strain having an overlap in the region on the chromosome that happens to be related to the activity is obtained. Because mutations occur randomly on various chromosome sites, it was impossible to duplicate specific sites on the Koji mold chromosome. In addition, in the case of chromosomal duplication strains obtained by using such mutations, it is also known that reversion mutations (ie, loss of chromosomal duplication) occur frequently due to recombination between homologous sequences between them. It has been.

On the other hand, the genetic recombination method is a method of breeding by incorporating the target DNA into the koji mold using transformation, and the size of the gene incorporated by the conventional genetic recombination method is usually 5 to 6 kb. . However, in order to actually obtain useful koji molds with high enzyme productivity as described above, in addition to the target gene region, the promoter region and terminator region of the gene, and in some cases, a marker for screening It is necessary to be included in the DNA region into which the gene is incorporated, and when these regions are combined, the region is usually a large region of 10 kb or more.

As described above, there is no technology that can cause a relatively large region to overlap at an arbitrary position on a chromosome.

Japanese Patent No. 4469014

As described above, in the conventional method using the mutation process described in Patent Document 1, in practice, mutation occurs randomly at various sites on chromosomes. It is impossible to overlap them. Further, when a genome duplication region is generated on the same chromosome by such a conventional method, a back mutation (ie, chromosomal duplication is lost) is caused by homologous recombination in the process of culturing the transformant. Therefore, in the invention described in Patent Document 1, a strain that does not undergo reverse mutation is obtained, which is considered to be because an overlapping genomic region exists on a chromosome different from the original chromosome. Actually, in Patent Document 1, it is quantitatively confirmed that the activity and expression level of genes contained in the genome duplication region are increased, but it is confirmed where the duplication genome is located on the chromosome. Not.

The present invention solves the above-mentioned problems and enables stable and systematic acquisition of Neisseria gonorrhoeae with new traits that were previously unacquirable based on a technique for overlapping arbitrary genomic regions on the Aspergillus chromosome. The purpose is to.

In the bacterium belonging to the genus Aspergillus, the present inventor arranged the sequence of a transformation marker (selection marker) gene such as a pyrG gene outside the 5 ′ and 3 ′ ends of a region so as to sandwich an arbitrary region in the chromosome. By producing homologous recombination via the repair function after double-strand breaks generated in the homologous region inside the gene, and such strains are transformed into the transformation marker. By selecting based on the above, it was found that a strain (transformed bacteria) in which duplication (having a genome duplication region) occurred in a partial region of the genome on the same chromosome in the targeted chromosomal region can be produced. The present invention has been completed.

That is, the present invention relates to the following aspects.
[Aspect 1]
A transformant of a bacterium belonging to the genus Aspergillus, wherein the transformant has a genome overlap region (overlap genome region) in tandem on the same chromosome.
[Aspect 2]
The transformant according to aspect 1, wherein a transformation marker gene is incorporated in a region sandwiched between genome overlapping regions.
[Aspect 3]
The transformed bacterium according to

embodiment

1 or 2, wherein the genome overlap region is 10 to several hundred kb.
[Aspect 4]
The transformed bacterium according to any one of aspects 1 to 3, wherein the bacterium belonging to the genus Aspergillus is Aspergillus oryzae or Aspergillus soya.
[Aspect 5]
The transformed bacterium according to aspect 4, wherein the genome overlap region is 500 kb or more and less than 700 kb of the SC003 region of chromosome 2 of Aspergillus oryzae.
[Aspect 6]
The transformed bacterium according to embodiment 5, wherein the genome overlap region is a region sandwiched between AO090003001035 to AO090003001214 in the SC003 region of chromosome 2 of Aspergillus oryzae, and does not include these end ORF regions.
[Aspect 7]
The transformant according to any one of embodiments 1 to 6, wherein the transformation marker gene is selected from the group consisting of pyrG, sC and niaD.
[Aspect 8]
As compared with a control strain (Ct strain) having no chromosomal duplication, the protease is increased by 1.9 times or more and / or the α-amylase activity is increased by 1.4 times or more. 8. The transformed bacterium according to any one of 7.
[Aspect 9]
The transformed bacterium according to any one of aspects 1 to 8, which is produced by a gene recombination method.
[Aspect 10]
A soy sauce produced using the transformed bacterium according to any one of aspects 1 to 9.

According to the present invention, it is possible to cause duplication of an arbitrary region ranging from 10 to several hundred kb (for example, 500 kb or more and less than 700 kb) on the same chromosome of Neisseria gonorrhoeae. There has been no such technique in the past. In addition, since the present invention uses only an endogenous gene in the host and is a self-cloning strain in which no foreign gene remains in the produced duplicate strain, microorganisms used for food production such as soy sauce brewing (for example, It is also an extremely excellent method for breeding soy sauce koji.

Furthermore, in a transformed bacterium having an overlap of a chromosomal region produced by the inventive method, when a genome overlap region is lost due to recombination between homologous sequences arranged in tandem, it is incorporated into a region sandwiched between these genome overlap regions. PyrG and other transformation marker genes are dropped together and become specific auxotrophic (for example, uridine requirement) properties. As a result, they cannot grow on normal media, so selective pressure is applied and chromosome duplication occurs. Can be avoided.

An outline of the method of the present invention is shown. The production of 5'ΔpyrG or 3'ΔpyrG units is shown. The target region of chromosomal duplication in chromosome 2 is shown. The result of confirmation of the genomic region on the same chromosome by PCR is shown. The number of copies of each gene in the overlapping region of chromosome 2 using quantitative PCR is shown.

The present invention relates to the above-mentioned transformed bacterium, which is a transformed bacterium belonging to the genus Aspergillus and has a genome overlap region in tandem on the same chromosome. One of the main characteristics of this transformed bacterium is that a transformation marker gene is incorporated in a region sandwiched between genome overlapping regions. As a result, in the transformed bacterium of the invention method, when the genome overlap region is lost due to recombination between homologous sequences arranged in tandem, pyrG or the like in which a transformation marker gene is incorporated in the region sandwiched between the genome overlap regions, etc. The transformation marker genes of the two are dropped together to have a specific nutritional requirement (for example, uridine requirement), and as a result, they cannot grow on a normal medium, so that selective pressure is applied and the loss of chromosome duplication is avoided. It can be done.

Examples of the bacteria belonging to the genus Aspergillus used as the parent strain of the transformant of the present invention include arbitrary strains such as Aspergillus soya, Aspergillus oryzae, Aspergillus niger, Aspergillus awamori, etc., of which Aspergillus soya and Aspergillus -A strain belonging to oryzae is preferred.

Examples of such strains include Aspergillus soja 262 (FERM P-2188), Aspergillus soya 2165 (FERM P-7280), Aspergillus soya moth (ATCC 42251), Aspergillus oryzae (IAM2638), and Aspergillus oryzae RIB40. Examples include each strain that is stored in a public depository such as (NBRC100599) and is readily available to those skilled in the art.

It is known that foreign DNA is integrated into the chromosome through a repair mechanism during double-strand breaks in the chromosomal DNA. This DNA repair mechanism includes homologous recombination and non-homologous recombination (non-homologous end joining). There are two types of mechanisms. In the case of homologous recombination, integration occurs through a region homologous to foreign DNA, but in the case of non-homologous recombination, integration occurs at a random position on the chromosome regardless of the sequence of the foreign DNA. These two recombination mechanisms are thought to act in equilibrium (Ristic et al. Nucl. Acids Res. (2003) 31: 5229-5237).

The genes that form the center of the homologous recombination mechanism are a series of genes called the rad52 group, which includes rad50, 51, 52, 54, Mre11, XRS2, etc. (Kooistra et al. 2004). The homologous recombination mechanism has been confirmed in many species from bacteria to eukaryotes, and the uvsC gene has been cloned and studied in Aspergillus nidulans, which is a laboratory strain of the genus Aspergillus and has mononuclear conidia. (van Heemst et al. Mol. Gen. Genet. (1997) 254: 654-64), it has been reported that homologous recombination frequency is improved by increasing the expression frequency (Natsume et al. Biosci). Biotechnol. Biochem. (2004) 68: 1649-1656).

On the other hand, it is clear that non-homologous recombination mechanism is based on non-homologous end joining (Non-Homologous End Joining), which is completely different from homologous recombination. The genes at the center of this mechanism are Ku70, Ku80, Xrcc4, LIG4, DNAPKcs, etc. are known. Ku70 and Ku80 function as heterodimers, forming a complex with nucleotide kinase (XRCC4) and DNA Ligase VI, and linking to the DNA ends for its repair during DNA double-strand breaks (DSB) and non-homomologous End It is known to promote Joining. (Walker et al. Nature (2001) 412: 607-614). The existence of this homologous recombination mechanism via Ku has been confirmed only in eukaryotes.

The genome overlap region in the transformant of the present invention can be arbitrarily selected. The size of the region is preferably 10 to several hundred kb. As a preferred example of such a genomic overlapping region, a genomic region in the SC003 region of chromosome 2 of Aspergillus oryzae containing a large number of genes encoding various industrially useful enzymes such as protease and α-amylase, for example, A genomic region of 500 kb or more and less than 700 kb. In particular, the genome overlap region includes a region sandwiched between AO090003001035 to AO090003001214 in the SC003 region of Aspergillus oryzae chromosome 2, and does not include the ORF region at these ends. Preferred examples of such transformed bacteria include the A-C-585k strain, the B-C-517k strain, and the B-C4-512k strain described in the following examples.

In the transformed bacterium of the present invention, the expression levels of various industrially useful enzymes are increased as compared with a control strain (Ct strain) having no chromosome duplication. For example, in an Aspergillus oryzae transformed bacterium, protease activity is 1.8 times or more, preferably 3.1 times or more, and / or α-amylase activity is 1.4 times or more, preferably 1.6 times or more. It is characterized by an increase.

In the transformed bacterium of the present invention, the genomic region to be duplicated can be arbitrarily selected, so that various known regions encoding useful enzymes are used as the duplicated target region, soy sauce brewing, etc. A koji mold (for example, a soy sauce koji mold) useful for food production can be obtained. Therefore, the present invention also relates to foods such as soy sauce produced using such transformed bacteria.

There is no restriction | limiting in particular in the preparation method and means of the transformed microbe of this invention. One preferred method is a gene recombination method using a transformant as shown below.

That is, the transformant used in the gene recombination method of the present invention has a coding region 5 outside of either the 5 ′ end or 3 ′ end of the target region on the gonococcal chromosome for duplication. A transformation marker gene in which either one of the 'end or the 3' end is deleted is incorporated, and a transformation marker gene in which a part of the other end is deleted outside the other end of the target region. The target region is sandwiched between two transformation marker genes each lacking a part of the 5 ′ or 3 ′ end of the coding region, and is in contact with the defective portion of the incorporated transformation marker gene (Each terminal region sequence having a defective portion) is located on the side opposite to the target region.

That is, a part of the 5 ′ end of the coding region (5 ′ end region consisting of a base sequence of an appropriate length) is located outside the 5 ′ end of the target region on the chromosome of the bacterium belonging to the genus Aspergillus for duplication. A defective transformation marker gene was incorporated, while a part of the 3 ′ end of the coding region was deleted outside a part of the 3 ′ end of the target region (a 3 ′ end region comprising a base sequence of an appropriate length). A transformation marker gene can be incorporated. Alternatively, a transformation marker gene in which a part of the 3 ′ end of the coding region is deleted outside the 5 ′ end of the target region on the chromosome of a bacterium belonging to the genus Aspergillus intended for duplication, while the target region 3 A transformation marker gene lacking a part of the 5 ′ end of the coding region may be incorporated outside the “end”.

In any case, in two transformation marker genes that sandwich the target region to be overlapped and each lacks a part of the 5 ′ or 3 ′ end of the coding region, the sequence in contact with the missing portion is It must be located on the opposite side of the target area.

By culturing a transformant having such a structural feature, the coding region of the transformation marker gene incorporated between the corresponding chromosomes and outside the 5 ′ end or 3 ′ end of the target region, respectively. Homologous recombination between the homologous sequence regions (shaded areas in FIGS. 1, 2 and 4) existing in the middle part of the DNA via the repair mechanism at the time of double strand breakage, and the result A strain in which the target region is overlapped is obtained. Here, the “corresponding chromosome”, as shown in the Examples, includes many genes encoding homologous chromosomes contained in the polynuclear of the transformant, for example, useful substances (for example, various enzymes) in each bacterium. It means a plurality of chromosomes such as 8 and 2 included.

Therefore, as a bacterium belonging to the genus Aspergillus used in the present invention, a transformed bacterium in which various genes involved in the heterologous recombination mechanism as described above are suppressed or deleted (Japanese Patent Laid-Open No. 2006-158269). It can also be used.

In addition, the transformation marker gene incorporated outside the 5 ′ and 3 ′ ends of the target region in the transformant has a length sufficient to efficiently cause homologous recombination, for example, several hundred bp or more, The coding region lacks the 5 ′ end or part of the 3 ′ end so that a base sequence of several hundred bp to several kb (eg, about 100 bp to about 2 kb) remains in the middle of the coding region. Is preferred. That is, the 5 ′ end or part of the 3 ′ end of the coding region incorporated outside the 5 ′ and 3 ′ ends of the target region of the chromosome of the bacterium belonging to the genus Aspergillus for duplication is missing 2 In any one of the transformation marker genes, it is preferable that the base sequence having the above-mentioned length existing in the middle part of the coding region of the original transformation marker gene remains in common. Accordingly, the length of the base sequence of the 5 ′ end or 3 ′ end region to be deleted in the coding region of the transformation marker gene can be appropriately determined by those skilled in the art according to the type and total length of the transformation marker gene used. I can do it. For example, when the pyrG gene is used, it is usually about 0.4 kb to about 1.4 kb, for example. Further, the lengths of the base sequences of the 5 'end or 3' end regions to be deleted need not be the same.

When such a transformant is cultured, in the strain in which the target region is duplicated by the homologous recombination, the full length of the transformation marker gene coding region is simultaneously constructed. As a result, it is based on the transformation marker gene. Depending on the trait, a strain in which the target region is duplicated can be selected from a strain in which the target region is not duplicated (ie, the full length of the transformation marker gene coding region has not been constructed).

There are no particular limitations on the transformation marker used in the present invention, but typical examples of marker genes that can be positively selected include pyrG, sC, and niaD. When these marker genes are used, It is possible to select a strain in which the target region is overlapped by a trait that compensates for auxotrophy (uridine requirement, sulfur utilization, and nitrate utilization). *

These marker genes are cultured in a medium containing a drug for selection, and the bacteria containing the marker gene are expressed as cytotoxic substances by the expression product of the marker gene contained therein. It can be used for negative selection by converting to a cell death.

Furthermore, a homologous sequence region existing in the middle part of the coding region of either or both of the transformation marker genes to be incorporated outside the 5 ′ and 3 ′ ends of the target region on the chromosome for duplication is previously added to the I Appropriate restriction enzyme recognition sites known to those skilled in the art such as -sceI, I-ceuI, PI-pspI and PI-sceI can be introduced. Such restriction enzyme sites can be introduced by any means known to those skilled in the art, such as homologous recombination.

Preferred examples of the gene recombination method for producing the transformant of the present invention using the above transformant are as follows. The outline of this method is as shown in FIG. That is, the above method
(1) culturing a transformant having the above characteristics;
(2) through a repair mechanism at the time of double-strand breaks between homologous sequence regions present in common in the middle part of the coding region of the transformation marker gene incorporated outside the 5 ′ end and 3 ′ end of the target region And obtaining a strain in which the target region is duplicated by homologous recombination between corresponding chromosomes of the transformant,
(3) selecting a strain in which the target region is duplicated by a trait based on the transformation marker gene in which the full length of the coding region has been constructed by the homologous recombination.

In this gene recombination method, by culturing the above-mentioned transformant under normal conditions, it is appropriate to break double strands between homologous sequence regions that are commonly present in the middle part of the coding region of the transformation marker gene. It utilizes homologous recombination via a repair mechanism that works when it occurs at a low frequency. In the above method, transformants are once converted into protoplasts by any method known to those skilled in the art, and are maintained under appropriate conditions for a certain period of time (for example, several tens of minutes to 1 hour), and then cultured. Can increase the efficiency of homologous recombination. In addition, gonococci such as Aspergillus sojae and Aspergillus oryzae always maintain a multinucleated state in their life cycle including the conidial state, so that the above homologous recombination easily occurs between corresponding chromosomes. Conceivable. On the other hand, other fungi of the genus Aspergillus, such as Aspergillus nidulans, niger, fumigatus, awamori, etc., have mononuclear generations, but even those fungi can easily obtain multinucleated protoplasts from mycelia. Can do.

Furthermore, a trait in which an appropriate restriction enzyme recognition site known to those skilled in the art such as I-sceI, I-ceuI, PI-pspI, and PI-sceI has been introduced in advance into a homologous sequence region present in the middle part of the coding region. When a conversion marker gene is used, homologous recombination can be caused by culturing the transformant under the action of the restriction enzyme. Specifically, for example, by mixing a transformant in a protoplast state with a restriction enzyme in the presence of a fusion aid (eg, PEG) (protoplast PEG method), the restriction enzyme is added to the transformant. It is possible to act efficiently.

In addition, said transformant can be produced using a well-known means of those skilled in the art as described in the Example of this-application specification. Further, the transformant can be cultured under appropriate conditions known to those skilled in the art.

EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, the technical scope of this invention is not limited to these Examples.

As described below, a gonococcal chromosome 2 duplicated strain was prepared.

[experimental method]
Strains used
Aspergillus oryzae RP-1 strain (ΔpyrG). The Aspergillus oryzae RP-1 strain is a pyrG deletion strain derived from RIB40 (= ATCC42149) (Takahashi et al. (2006) Biosci Biotechnol Biochem. 70: 135-143).

Using medium <br/> poly peptone dextrin (PD) medium (polypepton 1%, dextrin 2% , KH 2 PO 4 0.5%, NaNO 3 0.1%, MgSO 4 0.05%, casamino acid 0.1%, pH 6.0), CzapekDox (CZ ) 1.2M sorbitol CZ was used as the minimum medium and regeneration medium. A CZ medium plate containing 1.5 mg / ml 5fluoroortic acid (5FOA) (Sigma) and 20 mM uridine was used as a medium for positive selection of pyrG- (uridine requirement) strain.

Transformation
Conidia were inoculated into 50 ml of a polypeptone dextrin liquid medium containing 20 mM Uridine in a 150 ml Erlenmeyer flask, and cultured at 30 ° C. for about 20 hours with shaking to recover the cells. The collected cells were washed with 0.7 M KCl buffer, and gently shaken in 0.7 M KCl buffer containing 1% Lysing enzyme (Sigma) at 30 ° C. for 3 hours to prepare protoplasts. The obtained protoplast was washed with 1.2 M sorbitol buffer, and then transformed by the protoplast PEG method. Transformants were regenerated on 1.2M sorbitol-CZ medium containing 0.5% agar.

Production of Chromosomal Duplicate Strains Chromosome duplicated strains were produced as follows. After adding 20 μl of PEG solution to a protoplast solution of about 2 × 10 7/100 μl, hold in ice for 40 minutes, add ⁷⁰ μl of PEG solution, and then hold at room temperature for 20 minutes, then 1.2M sorbitol -Regenerated on CZ media plate. When I-sceI was used, it was added simultaneously with the addition of the PEG solution. Strains that grew on CZ medium were used as chromosome duplication candidate strains for the subsequent analysis.

Comparison of gene copy number in chromosome using quantitative PCR (real-time PCR) Quantitative PCR was performed using Mx3005P (Agilent Technology). By using rad52 located in another chromosome as a normalizer, the copy number of each gene of Alp, amyR, prtT, and 1258D on chromosome 2 was compared between the parent strain and the chromosome duplication strain by relative quantification. The PCR conditions were maintained at 95 ° C. for 10 minutes, and then 45 cycles of 95 ° C. 20 seconds-58 ° C. 30 seconds-72 ° C. 30 seconds were repeated. For PCR, Rad52, Alp, amyR, prtT, and 1258D genes were amplified using r52U-r52L, amyRU-amyRL, AlpU-AlpL, prtTU-prtTL, and 1258DU-1258DL primers, respectively (Table 1).

Preparation method of bran meal The enzyme activity of Aspergillus was evaluated according to a conventional method. That is, 5 g of wheat bran sprinkled with 80% water is put into a 150 ml Erlenmeyer flask, sterilized at 121 ° C. for 50 minutes, inoculated with about 2 platinum ears and cultured at 30 ° C. for 4 days. After culturing, add 100 ml of sterilized water, shake with rubber plugs, shake well, and let stand at room temperature for 4 hours, then filter the extract obtained by filtering through No. 2 filter paper (manufactured by Advantech). An enzyme sample was used.

Method for measuring protease activity The obtained enzyme sample was appropriately diluted and measured according to the method described in "Soy sauce test method" (Japan Soy Sauce Research Institute, 1985, p. 287). Protease activity is shown as 1 U (unit or unit) that produces 1 μmol of tyrosine per gram of wheat bran per gram.

Method for measuring α-amylase activity The obtained enzyme sample was appropriately diluted and measured using an α-amylase measurement kit (Kikkoman Brewing Analysis Kit, Code 60213) according to the protocol of the kit. The α-amylase activity was expressed as 1 U (unit or unit) of a titer that liberates 1 μmol of 2-chloro-4-nitrophenol per 1 g of bran koji.

[Creation of chromosome duplicated strain]
Chromosomal duplication is thought to occur in the process of chromosome double-strand break repair, but the mechanism is unknown. However, when analyzing the chromosomes of mutant strains obtained by gamma irradiation in yeast, it has been reported that there are repetitive sequences near the boundaries of the regions where chromosome duplication occurred (Non-patent Document 1). It is considered that chromosomal duplication can be produced by preparing a construct having the overlapping target region sandwiched between 5′ΔpyrG and 3′ΔpyrG possessed, and causing double-strand breaks within the homologous sequence (FIG. 1).

Therefore, first, in order to produce 5′ΔpyrG and 3′ΔpyrG, basic units as shown in FIG. 2 were produced. The 5'ΔpyrG or 3'ΔpyrG unit was prepared using PCR and ligation. The pyrG unit is prepared using a method known to those skilled in the art in FIG. 2 near the transcription start point of the full pyrG (in the case of 5′ΔpyrG) or near the 3 ′ side of the coding region (in the case of 3′ΔpyrG). This was carried out by incorporating a homologous sequence indicated by hatching (which may contain an appropriate restriction enzyme recognition site). After each vector is integrated on the chromosome, a 5FOA resistant strain is selected (negative selection), thereby obtaining a 5'ΔpyrG or 3'ΔpyrG strain whose interior is excised by recombination in the homologous region.

Next, a vector for integrating each unit of 5'ΔpyrG and 3'ΔpyrG into a target region on the chromosome was constructed. As shown in FIG. 3, the target region is a 700 kb region (FIG. 3, hatched portion) including a portion corresponding to each ORF region of AO090003001003 to AO090003001258 of SC003 region in chromosome 2 of A. oryzae. A 512-kb region including the portion corresponding to each ORF region of AO090003001036 to AO090003001212 from the B region to the C4 region (AO090003001035 to AO090003001214, not including these ORF regions) Area). In the present specification, for the sake of simplification, the AO090003000 portion of each gene name is omitted and expressed by B (eg, AO090003000160 → B160). The sequence of each gene corresponding to the SC003 region is based on DOGAN (Database Of the Genomes Analyzed at NITE), a genome analysis database of the National Institute of Technology and Evaluation (NITE). (Http://www.bio.nite.go.jp/dogan/GeneMap?GENOME_ID=ao__G2).

A vector (pB-5'Δ) for incorporating the 5′ΔpyrG construct into the B region on chromosome 2 in FIG. 3 and a vector (pC4-3′Δ) for incorporating the 3′ΔpyrG construct into the C4 region in FIG. Was constructed as follows.

First, using the primers B-U and B-L and C4-U and C4-L, a region of about 3 kb including the vicinity of the B region and the vicinity of the C4 region was amplified from the genomic DNA of A. oryzae by PCR and cloned into a vector.

A vector containing the vicinity of the B region amplified using primers B-iU and B-iL, and a 5′ΔpyrG unit amplified using primers pyrU and pyrL, were obtained using an In-fusion cloning kit (Takara). Ligation was carried out to construct a vector (pB-5′Δ) for incorporating a 5′ΔpyrG construct into the B region on chromosome 2. Ligation was performed according to the protocol specified in the kit. Subsequently, the vector containing the C4 region amplified using the primers C4-iU and C4-iL and the 3′ΔpyrG unit amplified using the primers pyrU and pyrL were obtained using an In-fusion cloning kit (Takara). Ligation was carried out to construct a vector (pC4-3′Δ) for incorporating a 3′ΔpyrG construct into the C4 region. The primers used to make this vector are shown in Table 2.

First, a vector (pB-5'Δ) for integrating the 5′ΔpyrG construct into the B region of chromosome 2 was amplified using primers BU and BL, and the PCR product was used to amplify the ΔpyrG strain of A. oryzae. Transformation. As a result of examining the obtained transformant by PCR and Southern hybridization, it was confirmed that the vector was integrated into the target site in one strain. Next, conidia were collected from this strain, applied to approximately 1 × 10 ⁵ onto a CZ medium plate containing 5FOA, and the obtained 5FOA resistant strain was analyzed. As a result, the B region had a 5′ΔpyrG construct. Was confirmed. A ΔpyrG strain having a 5′ΔpyrG construct in the B region is used as a parent strain, and a vector (pC4-3′Δ) for incorporating the 3′ΔpyrG construct in the C4 region (for 512 kb region duplication) is used as primers C4-U and After amplification by PCR using C4-L, transformation was performed. A strain in which the vector was incorporated into the C4 region was selected using PCR and Southern hybridization, and conidia were collected. As a result, colonies exhibiting resistance on the 5FOA-CZ plate were selected. The construction of a strain in which 5`ΔpyrG was incorporated into the B region, which is the most centromeric side, and 3`ΔpyrG was incorporated into the C4 region, which was the most telomeric side, was confirmed. Thus, a strain (B-C4-hap strain) having a basic construct for overlapping the 512 kb region in chromosome 2 was successfully constructed (FIG. 3).

Next, using this B-C4-hap strain as a parent strain, an experiment for obtaining a chromosome duplication strain was performed by the method described above. As a result, one pyrG + colony was obtained from the regenerated plate, and this was designated as B-C4-512k strain. Subsequently, chromosomal DNA was extracted from the obtained pyrG + strain (B-C4-512k strain), and an attempt was made to confirm whether the chromosomes were duplicated by PCR and quantitative PCR (real-time PCR).

Confirmation of chromosomal duplication by PCR First, it was confirmed by PCR whether or not duplication of the targeted chromosome region occurred. The result is shown in FIG. When using primers (BU and BL) that are located at the centromere end of the overlapping region and increase the region near the B region where the vector (pB-5'Δ) is incorporated (upper figure 4) A 4.4 kb band was amplified in both the control strain (parent strain: B-C4-hap strain) and the overlapping strain (B-C4-512k strain) (bottom of FIG. 4, lanes 1 and 2). In addition, when primers (C4-U and C4-L) are used to increase the region near the C4 region, which is located at the telomeric end of the overlapping region and into which the vector (pC4-3'Δ) has been incorporated (Fig. 4)), amplification of a band of 3.9 kb was observed in both the control and overlapping strains (bottom of FIG. 4, lanes 5 and 6). On the other hand, when PCR was performed with a combination of primers C4-U and BL (Fig. 4), no band amplification was observed in the control, whereas in the chromosome duplicated strain, a clearly 4.8 kb band was observed. Amplification was seen (lanes 3 and 4). The 4.8 kb band found only in the chromosomal overlapping strain is a band obtained only when the targeted region on the chromosome overlaps on the same chromosome and the structure shown in the lower part of FIG. 4 is present on the chromosome. it is conceivable that. From this, it was shown that the 512-kb region (B1036 to B1212) extending from the B region to the C4 region on chromosome 2 overlaps on the same chromosome in the chromosome overlapping strain B-C4-512k.

Confirmation of copy number by real-time PCR Subsequently, the copy number of each gene in the overlapping region of chromosome 2 was determined using real-time PCR (quantitative PCR) and the control strain (parent strain: B-C4-hap strain) and chromosome duplication The strain (B-C4-512k strain) was compared (FIG. 5). In the experiment, rad52, which is present in other chromosomes and has a copy number of 1, is used as a normalizer, and the B1036 gene (Alp) located at the centromere end in the overlap region is located 490 kb away from it. Relative quantification using SYBR Green targeting the B1208 (amyR) gene, the B1212 (prtT) gene located at the telomeric end, and the B1258D gene located outside the overlapping region, compared with the copy number in the parent strain Went. FIG. 5 shows relative values with respect to the copy number in the control strain (B-C4-hap strain). As a result, in the B-C4-512k strain, which is a chromosome duplication strain, the relative value of B1258D located outside the chromosome duplication region was about 1, but the B1036 gene, B1208 gene, B1212 located inside the duplication region were about 1 All genes showed values about twice the copy number in the control strain. From these results, it was confirmed that in the chromosome duplication strain (B-C4-512k strain), the copy number of the gene located in the duplication region was amplified to 2. From the results of these PCR and real-time PCR (quantitative PCR), it was confirmed that the 512 kb region, which was the target region on chromosome 2, was indeed duplicated in the chromosome duplication strain (B-C4-512k strain). It was done.

Further, in the same manner, 700 kb between AD regions in FIG. 3, 585 kb between AC regions, 517 kb between BC regions, 573 kb between A-C2 regions, and 10 kb between B-B3 regions In order to obtain a strain, a vector for introducing a 5′ΔpyrG construct into the A region and a 3′ΔpyrG construct into each of the B3 region, C2, C region, and D region was constructed. For vector construction, primers A, AL, A-iU and A-iL are used for the A region, primers B3-U, B3-L, B3-iU and B3-iL are used for the B3 region, and the C region. Includes primers CU, CL, C-iU, C-iL, C2 region includes primers C2-U, C2-L, C2-iU, C2-iL, D region includes primers DU, DL, D-iU D-iL was used, and ligation with the ΔpyrG unit was performed using an In-fusion cloning kit (Takara). After amplifying the obtained vector by PCR in the same manner as described above and then transforming it into the ΔpyrG strain of A. oryzae, a parent strain for chromosomal duplication was prepared, and then a duplicate strain of AD region 700 kb (AD-700k strain), AC region 585 kb overlapping strain (AC-585k strain), BC region 517 kb overlapping strain (BC-517k strain), A-C2 region 573 kb overlapping strain (A-C2-573k strain), A 10-kb overlapping strain (B-B3-10k strain) of the B-B3 region was obtained. Table 3 shows the primers used for the production of these overlapping strains. Regarding these strains, it was shown that each region on chromosome 2 was duplicated on the same chromosome as in the case of the chromosome duplication strain B-C4-512k.

Measurement of activity For duplicated strains of chromosome 2, refer to Patent Document 1 (Patent No. 4469014, “Koji mold that retains large-scale genome duplication”), and genomic region corresponding to AO090003001003 to AO090003001259 of SC003 on chromosome 2 It has been reported that a significant increase in protease activity and amylase activity can be seen in duplicate strains of. In order to investigate whether or not the same activity increase was observed for the duplicated strain in which chromosome 2 was duplicated in this experiment, enzyme activity was measured. As a comparative control of enzyme activity, the AD-700k strain in which the region of 700 kb corresponding to AO090003001003 to AO090003001258 (B1003 to B1258) on chromosome 2 was overlapped using the method in this experiment ( FIG. 3). Furthermore, enzyme activity was measured using duplicate strains AC-585k, BC-517k, A-C2-573k, and B-B2-10k produced by the same method.

Results of measurement of protease activity Using the above-mentioned method, total protease activity was measured using the enzyme extract obtained from bran rice cake, and control strains (Ct strains) that do not have chromosomal duplication and duplicate strains were measured. A comparison was made. The results are shown in Table 4. As for the protease activity, the activity value increased about 3 times in the chromosome duplication strain (A-C4-512k strain) compared to the activity of the control strain (Ct strain). This value was almost the same as the activity value obtained with the chromosome duplication strain (AD-700k strain) in the 700 kb region. Therefore, the 512 kb chromosomal duplication strain (B-C4-512k strain) prepared in the present invention has the same protease activity as the 700 kb chromosomal strain duplication (AD-700k strain) in bran culture. An increase was found. In addition, the same increase in protease activity was also confirmed in the 575 kb overlapping strain (AC-575k strain) and the 517 kb overlapping strain (BC-517k strain) between the BC regions prepared by the same method. On the other hand, in the 10-kb overlapping strain between B-B3 regions (B-B3-10k strain) and the 573-kb overlapping strain between A-C2 regions (A-C2-573k strain), the increase in protease activity was slightly less than twice. It was.

Results of measurement of α-amylase activity Subsequently, α-amylase activity was also measured using the same enzyme solution. The results are shown in Table 5. As a result, it was revealed that the activity value in the chromosome duplication strain (B-C4-512k strain) was increased by about 1.6 times that of the control strain (Ct strain). This value was almost the same as the value obtained in the chromosome duplication strain (AD-700k strain) of the 700 kb region. The same increase in amylase activity was also observed in chromosome duplication strains other than the B-B3-10k strain (AC-585k strain, BC-517k strain, A-C2-573k strain). Therefore, the chromosomal duplication strain (B-C4-512k strain) of the 512 kb region produced in the present invention has the same amylase activity in the bran culture as the chromosomal strain duplication (AD-700k strain) of the 700 kb region. An increase was found.

From the above results, in each transformed bacterium prepared in this example, an overlapping region was observed on the same chromosome of chromosome 2. For example, in the B-C4-512k strain, the 512 kb region between B1036-B1212 of chromosome 2 is surely duplicated on the same chromosome, and in the bran culture, a 700 kb chromosomal overlapping strain (B1003-B1258 region) As in the case of AD-700k strain), it was found that a remarkable increase in protease activity and amylase activity was observed as compared with the parent strain having no chromosome duplication. In addition, the same protease activity and amylase activity increased in the 575 kb overlapping strain between AC regions (AC-575k strain) and 517 kb overlapping strain between BC regions (BC-517k strain) prepared in the same manner. Was confirmed. From this, it was shown that the duplication of a region shorter than the region of 700 kb between B1003-B1258 is sufficient for the increase of protease activity and amylase activity due to the duplication of chromosome 2.

According to the present invention, a chromosomal region that is considered to be practically useful is identified using genomic information of a strain to be bred, and a strain obtained by overlapping the specific genomic region in tandem on the same chromosome is provided. It is possible to promote molecular breeding efficiently. Furthermore, it is expected that a koji mold with a completely new and useful character that has not been conventionally known can be obtained.

Claims

A transformant of a bacterium belonging to the genus Aspergillus, wherein the transformant has a genome overlap region in tandem on the same chromosome.
2. The transformed bacterium according to claim 1, wherein a transformation marker gene is incorporated in a region sandwiched between genome overlapping regions.
The transformed bacterium according to claim 1 or 2, wherein the genome overlap region is 10 to several hundred kb.
The transformed bacterium according to any one of claims 1 to 3, wherein the bacterium belonging to the genus Aspergillus is Aspergillus oryzae or Aspergillus soya.
The transformed bacterium according to claim 4, wherein the genome overlap region is 500 kb or more and less than 700 kb of the SC003 region of chromosome 2 of Aspergillus oryzae.
The transformed bacterium according to claim 5, wherein the genome overlap region is a region sandwiched between AO090003001035 to AO090003001214 in the SC003 region of Aspergillus oryzae chromosome 2, and does not include the ORF region at these ends.
The transformed bacterium according to any one of claims 1 to 6, wherein the transformation marker gene is selected from the group consisting of pyrG, sC and niaD.
The protease is increased by 1.9 times or more and / or the α-amylase activity is increased by 1.4 times or more compared with a control strain (Ct strain) having no chromosome duplication. The transformant as described in any one of thru | or 7.
The transformed bacterium according to any one of claims 1 to 8, which is produced by a gene recombination method.
Soy sauce produced using the transformant according to any one of claims 1 to 9.