JP2018064484A - Methods for mass-producing hydrolytic enzymes by aspergillus oryzae - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide transformants of Aspergillus oryzae that mass-produce hydrolytic enzymes such as protease, mannose and xylanase while maintaining high conidium productivity, and methods for mass-producing such enzymes by culturing the transformants.SOLUTION: The invention relates to a transformant of Aspergillus oryzae characterized in that a promoter of a hydrolytic enzyme gene is functionally linked to a transcription controlling region of a nucleic acid sequence encoding a transcription factor that regulates the expression of the hydrolytic enzyme gene. The invention also relates to a method comprising culturing the transformant for increasing the production or secretion amount of the hydrolytic enzyme without lowering the conidiogenesis in the transformant.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a transformant of Aspergillus oryzae in which the activity of a hydrolase is increased and a decrease in conidia-forming ability associated therewith is suppressed, a DNA cassette for transformation for producing the transformant, and And a method for increasing the activity of hydrolase in Neisseria gonorrhoeae, including culturing the transformant.

  Aspergillus sojae and Aspergillus oryzae are widely used industrially for brewing traditional foods such as soy sauce, sake, miso and enzyme production. Also, recent Aspergillus oryzae (Machida, M., Nature, 438, 1157-1161 (2005)) and Aspergillus soya (Atsushi Sato, et al., DNA Res. 2011 Jun; 18 (3): 165-176). With the progress of the determination of the whole genome sequence and comprehensive gene expression analysis using microarrays, genetic engineering modifications, especially chromosomal modification, are expected to improve the productivity and growth rate of enzymes, etc. It is a filamentous fungus.

  Such koji molds produce and secrete proteins; polysaccharides such as β-mannan, cellulose and xylan; lipids; and hydrolases that act in the degradation system of various types of substances such as starch, and are excellent in that. It is widely used in the production of brewed foods due to its ability to saccharify starch and its ability to degrade proteins.

  So far, for the purpose of controlling and increasing the expression of gonococcal hydrolase, etc., as described in Patent Document 1 and Patent Document 2 by the present applicant, Various transcription factors that control gene expression have been analyzed.

  Further, Patent Document 3 describes a nucleic acid cassette for overexpressing xylanase A in Aspergillus niger and the like. As an example of the nucleic acid cassette, the promoter of the enzyme is a regulator of the enzyme (xlnR). The structure used as a promoter of the nucleic acid sequence is described. Thus, a system that uses a promoter of a target gene activated by a transcription factor as a promoter of the transcription factor is generally called “positive feedback (loop)”.

  Patent Document 4 and Patent Document 5 also describe an expression system that can constitute such a “positive feedback” for the transcription factor prtT in a protease expression system such as Aspergillus niger.

  Furthermore, Patent Document 6 and Non-Patent Document 1 disclose a self-amplification cassette based on a positive feedback loop (positive feedback loop) capable of expressing an artificial transcription factor protein in an exogenous gene expression system in yeast. Have been described.

  However, in the prior art including the invention described in the above-mentioned literature, although mention is made regarding the increase in the production or activity of the target enzyme by the positive feedback function in transcription factor expression, There was no description or suggestion about the relationship between such a positive feedback function and the conidia formation ability.

Japanese Patent No. 5256402 Japanese Patent No. 5704609 Patent No. 4339400 Specification Japanese Patent No. 4563585 Special Table 2003-526374 Japanese Patent Laying-Open No. 2015-70806

A. Becskei et al. The EMBO Journal Vol. 20, no. 10, pp. 2528-2535, 2001

Therefore, the problem to be solved by the present invention is to transform a gonococcal transformant that produces a large amount of hydrolases such as protease, mannanase and xylanase while maintaining high conidia formation ability, and to culture the transformant It is to provide a method for producing the enzyme in large quantities.

  As a result of various studies, the present inventors have functionally placed the hydrolase gene promoter in the transcription control region of a nucleic acid sequence encoding a transcription factor that controls the hydrolase gene expression in Neisseria gonorrhoeae. As a result of ligation, the transformant of Aspergillus oryzae whose activity of hydrolase (expressed amount of hydrolase) is increased as compared with the parent strain Aspergillus while the decrease in conidia formation ability is significantly suppressed. As a result, the present invention was completed.

That is, this invention has the following aspects.
[Aspect 1]
A transformant of Aspergillus oryzae, in which the activity of hydrolase is increased and the decrease in conidia-forming ability is suppressed compared to the parent strain Aspergillus.
[Aspect 2]
The transformant according to aspect 1, wherein the hydrolase is a protein hydrolase or a saccharide hydrolase.
[Aspect 3]
The transformant according to aspect 1 or 2, wherein the hydrolase is selected from alkaline protease, β-mannanase, and xylanase.
[Aspect 4]
The transformant according to any one of aspects 1 to 3, wherein the conidia-forming ability is obtained by measuring the number of conidia in a bran seed pod.
[Aspect 5]
The transformant as described in any one of aspects 1-4 whose Aspergillus is Aspergillus soja or Aspergillus oryzae.
[Aspect 6]
Any one of Embodiments 1 to 5, wherein a promoter of the hydrolase gene is operably linked to a transcription control region of a nucleic acid sequence encoding a transcription factor that controls expression of the hydrolase gene. The transformant according to one item.
[Aspect 7]
The transformant according to aspect 6, wherein the transcription factor belongs to the Zn (II) Cys6 type.
[Aspect 8]
The combination of a hydrolase gene promoter and a transcription factor that controls the expression of the hydrolase gene is an alkaline protease gene (alp) or leucine aminopeptidase gene (lapI) promoter and PrtT that is a transcription factor for them. The β-mannanase gene (manG) or endoglucanase gene (eglB) promoter and ManR which is a transcription factor for them; and the xylanase gene (xynF1), β-xylosidase gene (xylA) or endoglucanase gene promoter and to them The transformant according to embodiment 7, which is selected from the group consisting of each combination of transcription factors XlnR.
[Aspect 9]
A DNA cassette for producing a transformant of Aspergillus according to any one of Embodiments 1 to 8, wherein at least a part of a nucleic acid sequence encoding a transcription factor that controls expression of a hydrolase gene of Aspergillus, and A DNA cassette for transforming Neisseria gonorrhoeae, comprising a promoter for the hydrolase gene, wherein the promoter is operably linked to a transcription control region of the transcription factor.
[Aspect 10]
Incubating the transformant of Aspergillus according to any one of aspects 1 to 8, and increasing the production amount or secretion amount of hydrolase while suppressing a decrease in the amount of conidia formed in the transformant How to make.
[Aspect 11]
A hydrolase, wherein the transformant of Aspergillus according to any one of claims 1 to 8 is cultured, and the hydrolase is collected from the medium after secreting the protein hydrolase into the medium. Production method.

  According to the present invention, by using positive feedback in a transcription factor expression system, the influence on the conidia formation ability is reduced, and the transcription factor is controlled (regulated) while maintaining a relatively high conidia formation ability. The activity of the hydrolase encoded by the target gene of interest (enzyme production) can be greatly improved.

  In addition, once the transcription factor starts to be expressed by positive feedback, the amount of transcription increases at a stretch. Therefore, even if the expression intensity of the promoter itself is low, a large amount of transcription factor can be efficiently expressed. As a result, even when using a promoter whose expression intensity (transcription activating effect) is weaker than a constitutively expressed promoter such as tef-1, transcription equivalent to or higher than tef-1 can be achieved by positive feedback. Activity can be demonstrated.

  Further, in the transformant of the present invention, the conidia formation ability is remarkable although the enzyme activity is increased as seen when the target gene is forcibly expressed using a constitutively expressed promoter such as tef-1. The phenomenon of declining was not observed.

  Furthermore, in the transformant of the present invention, since the original expression pattern of the transcription factor is maintained, the influence on the growth and conidia formation of the koji mold can be reduced.

The outline of the transformation DNA cassette for forced expression of the transcription factor of the present invention and transformation by homologous recombination are shown. The analysis result of the transcription factor prtT forced expression strain of protease and peptidase is shown. The analysis result of the transcription factor manR forced expression strain of β-mannanase is shown. The analysis result of the transcription factor xlnR forced expression strain of xylanase is shown.

  The present invention relates to a gonococcal transformant (transformed bacterium) in which the activity of a hydrolase is increased and the decrease in conidia-forming ability is suppressed as compared to the parent strain gonococcus.

  Here, “gonococcus” is a general term for microorganisms (fungi) belonging to the genus Aspergillus. As a representative example, in addition to the already described Aspergillus oryzae and Aspergillus soya, it is used particularly in the food and brewing fields. Examples of the microorganisms that can be used include Aspergillus luchuensis. Moreover, substantially the same effect can be obtained even when industrially useful filamentous fungi used in food, brewing, chemistry, and medical fields other than koji molds are used. These bacteria can also be obtained as commercial products or from various public deposit institutions such as American Type Culture Collection (ATCC).

  Examples of the bacterium belonging to Aspergillus used as the parent strain (host) of the transformant of the present invention include arbitrary bacteria such as Aspergillus sojae, Aspergillus oryzae, Aspergillus leuchuensis, etc. Among them, Aspergillus soya and Aspergillus -A strain belonging to oryzae is preferred.

  Examples of such strains include Aspergillus soya 262 (FERM P-2188), Aspergillus soya 2165 (FERM P-7280), Aspergillus soya (ATCC 42251), Aspergillus soya (NBRC4239), Aspergillus oryzae (IAM2638). ), And Aspergillus oryzae RIB40 (deposit organization: Incorporated Administrative Agency Liquor Research Institute, deposit number ID: RIB40) and the like, each of which is easily available to those skilled in the art A strain etc. can be mentioned. The genome sequence of Aspergillus oryzae RIB40 strain was determined in 2005 (Nature, 438: 1157, 2005), and its information is available from the DOGAN database (http: //www.bio.nite). .go.jp / dogan / MicroTop? GENOME_ID = ao).

  Furthermore, as a suitable koji mold for producing the transformant of the present invention, genes involved in heterologous recombination mechanisms such as ku70, ku80 and ligD, which are also used in the examples of the present specification, are suppressed. Alternatively, a transformed bacterium having improved homologous recombination frequency due to deletion (disruption) or the like (Japanese Patent Laid-Open No. 2006-158269, Takahashi T, Masuda T, & Koyama Y, Biosci. Biotechnol. Biochem. 70 (2006)). 135-143; Takahashi T, Masuda T, & Koyama Y (2006), Mol Genet Genomics 275: 460-470; Maruyama J & Kitamoto K (2008), Biotechnol 30 : 1811-1817).

  Furthermore, it is preferable to use an appropriate auxotrophic strain known to those skilled in the art as the parent strain so that the target transformant can be easily isolated.

  The term “conidia” generally refers to a spore produced by ascomycetes and basidiomycetes as a means of asexual reproduction and sexual generations such as Aspergillus sojae and Aspergillus oryzae. Means spores formed by imperfect bacteria.

  As already mentioned, Neisseria gonorrhoeae produces and secretes proteins; polysaccharides such as β-mannan, cellulose and xylan; lipids; and hydrolases that act in the degradation system of various types of substances such as starch. This is well known to those skilled in the art. The hydrolases in the present invention include any of such hydrolases known to those skilled in the art.

  Among these, a protein hydrolase is also called a protease, and is a general term including proteinases that mainly degrade proteins such as alkaline protease, or peptidases that degrade small peptides such as endopeptidase and leucine aminopeptidase. Examples of the saccharide hydrolase include β-mannanase, cellulase, endoglucanase, β-xylosidase, xylanase and the like.

  “The hydrolase activity is increased compared to the parent strain koji mold” means that the hydrolase activity (hydrolase) expressed by the transformant of the present invention when compared under any of the same conditions. Production or secretion amount) is significantly increased (improved) compared to that of Aspergillus used as a host in producing the transformant, for example, several times to several times as compared with Aspergillus as the parent strain It means ten times, more specifically, an increase from about 2 to about 40 times. The activity of the hydrolase can be measured by any method / means known to those skilled in the art. For example, as described in the Examples of the present specification, after the conidia of Aspergillus oryzae grown on a Marz agar medium are statically cultured under certain conditions, the filtrate obtained from the culture is used as a crude enzyme solution ( Stock solution) is preferably used as a sample for measurement.

  Similarly, “decrease in the ability to form conidia is suppressed as compared with the parent strain koji mold” means that when the transformant of the present invention is produced when compared under any of the same conditions, It means that the degree of decrease (decrease rate) of the conidia forming ability of the transformant of the present invention is significantly suppressed as compared to the conidia forming ability (conidia formation amount) of the koji mold used.

  As described in the examples of the present specification, the conidia-forming ability (conidia-forming amount) is determined by measuring the number of conidia in a seed pod, in particular, a bran varieties. The body means that, for example, about 50% or more, preferably about 80% or more is maintained when the conidia forming ability of the parent strain Neisseria gonorrhoeae is 100%. Furthermore, as shown in the Examples of the present specification, some of the transformants of the present invention have an increased conidia-forming ability of about several tens of percent compared to the parent strain Neisseria gonorrhoeae.

  As a more specific embodiment of the transformant of the present invention, a promoter of the hydrolase gene is included in the transcription control region of a nucleic acid sequence (ORF) encoding a transcription factor that controls (activates) the expression of the hydrolase gene. Can be mentioned as a functionally linked transformant.

Here, “transcription factor” refers to a protein required in addition to RNA polymerase in a gene transcription reaction. Basic transcription factor, transcription initiation factor, transcription elongation factor, transcription termination factor, and transcription essential for transcription initiation. Including regulatory factors and the like, it particularly means protein factors involved in transcription initiation. A transcription factor binds to a promoter located upstream of the coding region of the gene to regulate and control the transcription reaction. Thus, since a transcription factor that is a protein has a function of interacting with DNA, a “Zn finger motif” (C ₂ H ₂ class, Cys _x class), Four types of "helix-turn-helix motif", "leucine zipper motif" and "helix-loop-helix motif" are known.

  Here, the “promoter” refers to a region of DNA involved in the efficiency of the transcription initiation reaction, and in a narrow sense, usually a region of about 40 base pairs before and after the transcription initiation position. This region includes an RNA polymerase binding site, a transcription factor binding site, and the like. Incidentally, the “promoter” in the present invention includes a nucleic acid sequence substantially lacking a part of the nucleic acid sequence of the promoter but substantially having a function as the promoter.

  Furthermore, the “transcriptional control region” is a sequence on DNA that is upstream of the coding region of a gene and controls (regulates) the transcription efficiency of the gene. In addition to the promoter, transcriptional regulation of enhancers, silencers, etc. Factors are included.

  Accordingly, “functionally linked” in the transformant of the present invention means that the hydrolase gene promoter linked to the transcription control region of the nucleic acid sequence encoding the transcription factor for the hydrolase gene (forced) The transcription factor itself binds to the transcription factor binding site contained in the expression promoter), and as a result, the expression of the transcription factor replaces the original promoter that controls the expression of the transcription factor. It means that it is controlled by the promoter of the hydrolase gene that is the control target (target gene). By such a mechanism, a “positive feedback” is formed in the expression system of the transcription factor in the transformant of the present invention. As a result, the transcription factor is forcibly expressed and the expression is controlled by the transcription factor (activity) It is thought that the expression level of the hydrolase gene increases.

  In such a transformant, the original promoter of the transcription factor does not need to be completely replaced with the promoter of the hydrolase gene, and at least a partial sequence of the original promoter of the transcription factor is It may be left as it is. However, even in such a case, the original promoter sequence of the transcription factor left in this way can no longer function as a promoter because of positional or structural reasons.

As a suitable example of a combination of a hydrolase gene that constitutes positive feedback in such a transformant and a transcription factor that regulates (activates) the expression of the hydrolase gene, an alkaline protease gene (alp) Or leucine aminopeptidase gene (lapI) and transcription factor PrtT; β-mannanase gene (manG) or endoglucanase gene (eglB) and transcription factor ManR for them; and xylanase gene (xynF1), β -Xylosidase gene (xylA) or each combination of endoglucanase gene and XlnR which is a transcription factor for them. All of these transcription factors are of the “Zn (II) Cys6” type belonging to the Cys _x class of the “Zn finger motif” already mentioned.

  Accordingly, the nucleic acid sequence of the promoter of the hydrolase gene that is the control target of the transcription factor is known to those skilled in the art from various koji molds as described above as preferred examples for producing the transformant of the present invention. It can be prepared by any method. The nucleic acid sequence of the hydrolase gene promoter is preferably obtained from the same strain as the host used in producing the transformant of the present invention, but can also be obtained from other strains.

  For example, these bacterial cells are cultured by the method described in the examples of the present specification, DNA is extracted by a conventional method, and the DNA thus obtained is used as a template and shown in Table 1 of the present specification. By a PCR reaction using each primer pair, a DNA fragment containing each nucleic acid sequence such as lapI promoter, manG promoter, eglB promoter, xynF1 promoter and xylA promoter can be amplified. The nucleic acid sequence of the DNA fragment thus obtained can be determined using a commercially available reagent and a DNA sequencer by any method known to those skilled in the art, if necessary. In addition to the nucleic acid sequence of each promoter, the above DNA fragment may contain other nucleic acid sequences such as homologous sequences for Fusion PCR.

  The PCR primer pairs for amplification of each promoter described in Table 1 are, for example, known information on the nucleic acid sequences of the above promoters (Kitamoto N1, Matsui J, Kawai Y, Kato A, Yoshi S, Ohmiya K , Tsukagoshi N., Appl Microbiol Biotechnol. 1998 Jul; 50 (1): 85-92.) And the like can be easily designed and prepared by those skilled in the art.

  Furthermore, a DNA fragment containing each nucleic acid sequence such as the promoter can be obtained by any method known to those skilled in the art, for example, a selection method by hybridization.

  The promoter used in the present invention is 90% or more, preferably 95% or more, more preferably 98% or more, and most preferably 99% with the nucleic acid sequence (base sequence) of each promoter obtained by the PCR reaction described above. A nucleic acid sequence (DNA) having a sequence identity of at least% and having a structure / function substantially equivalent to that of each corresponding promoter is also included.

  In order to determine the sequence identity between two nucleic acid sequences, the sequences are pre-processed optimally for comparison. For example, by making a gap in one sequence, the alignment with the other sequence is optimized. Thereafter, the bases at each site are compared. When the same base as the corresponding site in the second sequence is present at a site in the first sequence, the sequences are identical at that site. Sequence identity in the two sequences is expressed as a percentage of the total number of sites (total bases) of the number of sites that are identical between the sequences.

  In accordance with the above principles, the sequence identity between two base sequences is determined by the algorithm of Karlin and Altshul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990 and Proc. Natl. Acad. Sci. USA 90: 5873. -5877, 1993). A BLAST program using such an algorithm was developed by Altshul et al. (J. Mol. Biol. 215: 403-410, 1990). Further, Gapped BLAST is a program for determining sequence identity with higher sensitivity than BLAST (Nucleic Acids Res. 25: 3389-3402, 1997). The above program is mainly used for searching a database for sequences showing high sequence identity with a given sequence. These are available, for example, on the Internet website of National Center for Biotechnology Information.

  As sequence identity between sequences, Tatiana A. et al. The values determined using BLAST 2 Sequences software developed by Tatusova et al. (FEMS Microbiol Lett., 174: 247-250, 1999) are used. This software is available on the Internet website of National Center for Biotechnology Information. The programs and parameters used are as follows. In the case of a base sequence, the blastn program is used, and parameters are as follows: Reward for a match: 1, Penalty for amismatch: -2, Strand option: Both strands, Open gap: 5 and extension gap: 2 pen. : 10, word size: 11, Filter: ON is used. All parameters are used as default values on the website.

  Furthermore, the promoter used in the present invention is a nucleic acid sequence (DNA) that hybridizes under stringent conditions with the nucleic acid sequence (base sequence) of each promoter obtained by the above PCR reaction, and each corresponding promoter Also included are nucleic acid sequences having substantially the same structure and function as

  Here, stringent conditions are conditions in which a specific hybrid signal is clearly distinguished from a non-specific hybrid signal, and the hybridization system used, the type, sequence and length of the probe. It depends on. Conditions can be determined by changing the hybridization temperature, washing temperature and salt concentration. For example, when a non-specific hybrid signal is strongly detected, the specificity can be increased by raising the hybridization and washing temperature and, if necessary, lowering the washing salt concentration.

  If no specific hybrid signal is detected, the hybrid can be stabilized by lowering the hybridization and washing temperatures and, if necessary, raising the washing salt concentration. Such optimization can be easily performed by researchers in this technical field.

  As a specific example of stringent conditions, for example, hybridization is 5 × SSC, 1.0% (W / V) nucleic acid hybridization blocking reagent (Roche Diagnostics), 0.1% ( W / V) N-lauroyl sarcosine, 0.02% (W / V) SDS is used overnight (about 8 to 16 hours), and washing is performed at 0.5 × SSC, 0.1% (W / V). ) Using SDS, preferably 0.1 × SSC, 0.1% (W / V) SDS, twice for 15 minutes. The temperature for hybridization and washing is 52 ° C. or higher, preferably 57 ° C. or higher, more preferably 62 ° C. or higher, and most preferably 67 ° C. or higher.

  The transformant of Neisseria gonorrhoeae according to the present invention can be obtained by using a suitable genetic engineering means known to those skilled in the art, for example, a transcription factor that controls the expression of the hydrolase gene of Neisseria gonorrhoeae as shown in FIG. Including at least a part of the encoding nucleic acid sequence (functioning as a terminal sequence necessary for homologous recombination) and a promoter of the hydrolase gene, and the promoter is operably linked to the transcriptional control region of the transcription factor It is possible to prepare a DNA cassette for transformation characterized by the above, and transform the parent strain Koji mold based on homologous recombination using the cassette.

  The transformation DNA cassette includes any marker gene known to those skilled in the art to enable selection of transformed cells, at least a partial sequence of the original promoter of the transcription factor (homologous recombination). And other various elements such as a homologous sequence for Fusion PCR. Examples of the marker gene include genes that complement the auxotrophy of the host, such as URA3 and niaD, or resistance genes to drugs such as ampicillin, kanamycin, and oligomycin.

The transformation using the DNA cassette for transformation can be performed by a known appropriate method. For example, a method using polyethylene glycol and calcium chloride after protoplastization (Mol. Gen. Genet., 218, 99-104 (1989)) can be used.
The body can be mentioned.

  By culturing the transformant of Aspergillus oryzae according to the present invention by any method known to those skilled in the art, the production amount or secretion amount of hydrolase is increased while suppressing the decrease in the amount of conidia formed in the transformant. Can be made.

  Alternatively, the transformant of Neisseria gonorrhoeae according to the present invention is cultured by any method known to those skilled in the art, and after hydrolase is collected from the medium after secreting the protein hydrolase into the medium, the hydrolase Can be efficiently produced in large quantities.

  For example, the transformant of Aspergillus oryzae of the present invention can be cultured in a solid medium or liquid medium to secrete hydrolase into the medium, and then hydrolase can be collected from the medium. In such a production method, culture conditions such as culture system / medium selection, culture temperature / time, and the like can be appropriately set by those skilled in the art with reference to the following examples.

  In addition, a protein degradation product can be produced by mixing a culture obtained by culturing the transformant of Aspergillus of the present invention and a protein-containing raw material, and decomposing the protein in the raw material. There are no particular restrictions on the type of protein contained in the raw material used in this method, and examples include gelatin, collagen, and gluten. Proteolytic products of raw materials containing gelatin, collagen and gluten are useful as seasonings.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, the technical scope of this invention is not limited by a following example.

Production of various transcription factor forced expression strains (transformants) [Use medium]
Polypeptone dextrin (PD) medium; polypepton 1%, dextrin 2%, KH ₂ PO ₄ 0.5%, NaNO ₃ 0.1%, MgSO ₄ 0.05%, casamino acids 0.1%, pH 6.0, Neisseria gonorrhoeae Depending on the auxotrophy of the strain, Uridine was added to a final concentration of 10 mM.
Czapek-Dox (CD) minimal medium; Czapek Dox Broth (Japan BD, Cat. # 233810) 3.5%, agar 1.5% (0.5% for the overlay), pH 6.0, transformant During regeneration, sorbitol was added to 1.2M.
Malt's agar medium: malt extract 8%, agar 1.5%, pH 6.0.

[Preparation of DNA cassette for transformation]
FIG. 1 shows a DNA cassette for transformation for forced expression of transcription factors of Aspergillus soya and Aspergillus oryzae and an outline of transformation by homologous recombination. Genomic DNA was extracted from Aspergillus soya (NBRC4239 strain) and Aspergillus oryzae (RIB40 strain) according to a conventional method, and each DNA fragment was amplified by PCR using the primer set shown in Table 1 as a template. For PCR, KOD plus Neo (TOYOBO) was used. The amplified DNA fragment was purified using Gel Extraction Kit (QIAGEN). Fusion PCR was performed using the purified DNA fragment, and nested PCR was performed using the obtained PCR product as a template to prepare the DNA cassette for transformation of the present invention.

[Transformation of Aspergillus oryzae]
As a host (parent strain) for producing transformants of Aspergillus soya and Aspergillus oryzae, an auxotrophic strain that cannot grow on a minimal medium containing no uridine or uracil because of a mutation in the pyrG gene was used. . Since these strains also lack the ku70 gene that functions for non-homologous end joining, homologous recombination of the genome occurs with high efficiency (Takahashi T, Masuda T, & Koyama Y, Biosci. Biotechnol. Biochem. 70 (2006)). 135-143). When a DNA cassette containing a pyrG marker gene is introduced into these strains, it becomes possible to grow even in a minimal medium that does not contain uridine or uracil, so that the target transformant can be easily isolated. it can.

  Specifically, transformation was performed as described in the experimental procedure described below. 100 mL of PD medium containing uridine having a final concentration of 10 mM was added to a 500 mL baffled Erlenmeyer flask, and after conidia of the transformed host was ingested, the mixture was cultured with shaking at 30 ° C. for 16 hours. The cells in the culture solution were recovered using miracloth (Merck Millipore), washed with 0.6 M KCl buffer (pH 5.5), then 1.5% cellulase Onozuka (Yakult), 0.3% Yatalase. (Ozeki), protoplasts were prepared by suspending cells in 0.6 M KCl buffer containing 1.5% Lysing Enzyme (SIGMA) and culturing gently at 30 ° C. for 3 hours. Protoplasts were washed with 0.6 M KCl buffer and 1.2 M sorbitol buffer (pH 7.5), and a DNA cassette was introduced by the protoplast-PEG method for transformation. The transformant was inoculated in a CD minimal medium containing 1.2 M sorbitol, and the conidia of the obtained colonies were subcultured three times in a CD minimal medium not containing sorbitol to purify the nucleus. Homologous recombination is occurring as intended by amplifying the genomic sequence around the DNA cassette insertion region using the transformant genomic DNA as a template and subjecting it to agarose electrophoresis to analyze the length of the amplified fragment I confirmed.

Measurement of hydrolase activity [Koji mold culture method and preparation of crude enzyme solution]
The defatted soybean, wheat and water were mixed at a ratio of 1: 1.1: 1.5, 10 g was weighed into a 150 mL Erlenmeyer flask, put on a cotton plug and sterilized by an autoclave. To this, 1 platinum loop (about 1 × 10 ⁵ ) conidia grown on a Marz agar medium was inoculated, mixed, and then statically cultured at 30 ° C. for 72 hours. 100 mL of distilled water was added and stirred well. After stirring for 3 hours at room temperature with stirring every hour, No. It filtered with 2 filter papers (ADVANTEC). The obtained filtrate was used as a crude enzyme solution (stock solution).

[Measurement of alkaline protease activity]
1 mM N-Succinyl-Ala-Ala-Pro-Phe pNA (Suc-AAPF-pNA, SIGMA) was used as a substrate. The substrate solution was prepared by dissolving 25 mg of Suc-AAPF-pNA in 2 mL of DMSO and diluting 20 times with 20 mM Tris-HCl buffer (pH 8.0). 180 μL of the substrate solution and 20 μL of the enzyme solution obtained by appropriately diluting the crude enzyme solution with a buffer solution were mixed and reacted at 30 ° C. for 20 minutes, and then 600 μL of 0.1N HCl solution was added to stop the reaction. The blank was mixed in the order of enzyme solution, reaction stop solution, and substrate solution. The absorbance at 410 nm of the reaction solution and the blank solution was measured, and the enzyme activity was calculated from the difference in absorbance. The enzyme titer for producing 1 μmol of p-NA per minute under the above reaction conditions was 1 U.

[Measurement of β-mannanase activity]
Azo-carob galactomannan (Megazyme) was dissolved in 200 mM sodium acetate buffer (pH 4.0) to 2% to obtain a substrate. 150 μL of the crude enzyme solution was heated at 40 ° C. for 5 minutes, 150 μL of the substrate solution heated in the same manner was added, and reacted for 10 minutes. Only the weakly active control strain was incubated for 60 minutes. The reaction was stopped by adding 750 μL of 100% ethanol, and after centrifugation at 13,000 rpm for 10 minutes, the absorbance at 590 nm of the supernatant was measured. The blank was added to the crude enzyme solution in the order of the reaction stop solution and the substrate solution. The enzyme titer that produces 1 μmol of mannose per minute under the above reaction conditions was 1 U.

[Measurement of xylanase activity]
500 μL of the enzyme solution obtained by appropriately diluting the crude enzyme solution with 25 mM sodium acetate buffer (pH 4.7) was heated at 40 ° C. for 5 minutes, and then 1 Xylazyme AX tablet (Megazyme) was added. Reacted for 1 minute. The reaction was stopped by adding 10 mL of 2% Tris aqueous solution (pH> 9), and the mixture was allowed to stand at room temperature for 5 minutes. It filtered with 2 filter papers (Whatman). The absorbance at 590 nm of the filtrate was measured. The blank was added to the enzyme solution in the order of the reaction stop solution and the substrate tablet. The enzyme titer for producing 1 μmol of xylose per minute under the above reaction conditions was 1 U.

Measurement of conidia formation amount in bran seed bran Wheat bran and water were mixed at 1: 0.8, 5 g was weighed into a 150 mL Erlenmeyer flask, capped and sterilized by autoclave. This was inoculated with 1 platinum loop (approximately 1 × 10 ⁵ ) of conidia grown on a Marz agar medium, mixed, and then incubated at 30 ° C. for 72 to 96 hours. After stirring the bran seed bran after culturing, 0.3 g was weighed and 30 mL of distilled water was added and stirred well. Filtration was performed using a 70 μm cell strainer (Corning, Cat. # 352350), and the number of conidia contained in the filtrate was measured using a hemocytometer.

Results of Analysis of Protease and Peptidase Transcription Factor prtT Forced Expression Strain Using Aspergillus soya as a host, the translation elongation factor 1 (tef1) and histone H3 gene promoters exhibiting a constant expression pattern, and the PrtT activated promoter A prtT forced expression strain was prepared using a promoter of a host-derived leucine aminopeptidase gene (lapI) (Ptefl-prtT_OE strain, PhistoneH3-prtT_OE strain, PlapI-prtT_OE strain). As a result of preparing soy sauce cake using each forced expression strain and measuring the alkaline protease activity of the obtained crude enzyme solution (FIG. 2), the Ptef1-prtT_OE strain, the PhistoneH3-prtT_OE strain, and the PlapI-prtT_OE strain were control strains The activity was 2.3 times, 1.8 times and 2.5 times that of (parent strain). On the other hand, the number of conidia in the bran seed bran was 19%, 31% and 61% of the control strain, respectively.

Results of analysis of mannanase transcription factor manR forced expression strain Using aspergillus soya as a host, tef1 and the promoters of β-mannanase (manG) and endoglucanase (eglB) genes derived from the host activated by ManR ManR forced expression strains were prepared (Ptefl-manR_OE strain, PmanG-manR_OE strain, PeglB-manR_OE strain). Soy sauce cake was prepared using each forced expression strain, and β-mannanase activity of the obtained crude enzyme solution was measured (FIG. 3). The Ptef1-manR_OE strain, the PmanG-manR_OE strain, and the PeglB-manR_OE strain were respectively controlled. The activity was 41 times, 46 times and 30 times that of the strain (parent strain). On the other hand, the number of conidia in the bran seed bran was 0.05%, 119%, and 92% of the control strain, respectively.

Results of analysis of xylanase transcription factor xlnR forced expression strain Using aspergillus soya as the host, tef1, and the xylanase gene (xynF1) and eglB gene promoters derived from the host activated by XlnR, (Ptef1-xlnR_OE strain, PxynF1-xlnR_OE strain, PeglB-xlnR_OE strain). As a result of preparing soy sauce cake using each forced expression strain and measuring the xylanase activity of the obtained crude enzyme solution (upper figure), the Ptef1-xlnR_OE strain, the PxynF1-xlnR_OE strain, and the PeglB-xlnR_OE strain are control strains, respectively. The activity was 3.8 times, 7.5 times, and 5.6 times that of the parent strain. On the other hand, the number of conidia in the bran seed bran was 0.7%, 50% and 54% of the control strain, respectively.

  Similarly, using Aspergillus oryzae as a host, an xlnR forced expression strain was prepared using tef1 and the promoter of the host-derived xynF1 and β-xylosidase gene (xylA) activated by XlnR (Ptef1-xlnR_OE strain) , PxynF1-xlnR_OE strain, PxylA-xlnR_OE strain). As a result of preparing soy sauce cake using each forced expression strain and measuring the xylanase activity of the obtained crude enzyme solution (bottom of FIG. 4), the Ptef1-xlnR_OE strain, the PxynF1-xlnR_OE strain, and the PxylA-xlnR_OE strain are control strains, respectively. 1.9 times, 14 times, and 11 times the activity. On the other hand, the number of conidia in the bran seed bran was 0%, 88% and 79% of the control strain (parent strain), respectively.

  From the above results, when using a promoter of a gene showing a constant expression pattern such as tef1 or histoneH3 when forcibly expressing a transcription factor of Zn (IICys6 type) that controls the expression of gonococci hydrolase, It became clear that the number of conidia was significantly reduced.

  On the other hand, when a forcible expression strain of the transcription factor is produced using a hydrolase gene promoter activated by a transcription factor, the enzyme activity is equal to or higher than that of a strain using the tef1 gene promoter. At the same time, it was found that the decrease in conidia-forming ability relative to the parent strain was significantly suppressed, indicating higher conidia-forming ability than the forced expression strain using the tef1 promoter.

  From the above results, the expression of the hydrolase gene can be suppressed while minimizing the influence on the conidia formation system by forcibly expressing the transcription factor using the positive feedback mechanism of the transformant of the present invention. It was shown that it can be efficiently promoted.

  INDUSTRIAL APPLICABILITY The present invention is extremely important and useful industrially in the production of foods for seasonings, the production of pharmaceuticals such as digestives, and the production of protein hydrolase for detergents.

Claims (11)

A transformant of Aspergillus oryzae, in which the activity of hydrolase is increased and the decrease in conidia-forming ability is suppressed compared to the parent strain Aspergillus. The transformant according to claim 1, wherein the hydrolase is a protein hydrolase or a saccharide hydrolase. The transformant according to claim 1 or 2, wherein the hydrolase is selected from alkaline protease, β-mannanase, and xylanase. The transformant according to any one of claims 1 to 3, wherein the conidia-forming ability is obtained by measuring the number of conidia in a bran seed pod. The transformant as described in any one of Claims 1-4 whose Aspergillus is Aspergillus soja or Aspergillus oryzae. The promoter of the hydrolase gene is operably linked to a transcription control region of a nucleic acid sequence encoding a transcription factor that controls the expression of the hydrolase gene. The transformant according to claim 1. The transformant according to claim 6, wherein the transcription factor belongs to the Zn (II) Cys6 type. The combination of a hydrolase gene promoter and a transcription factor that controls the expression of the hydrolase gene is an alkaline protease gene (alp) or leucine aminopeptidase gene (lapI) promoter and PrtT that is a transcription factor for them. The β-mannanase gene (manG) or endoglucanase gene (eglB) promoter and ManR which is a transcription factor for them; and the xylanase gene (xynF1), β-xylosidase gene (xylA) or endoglucanase gene promoter and to them The transformant according to claim 7, wherein the transformant is selected from the group consisting of each combination of transcription factors XlnR. A DNA cassette for producing the gonococcal transformant according to any one of claims 1 to 8, wherein at least a part of a nucleic acid sequence encoding a transcription factor that controls expression of a hydrolase gene of gonococcus, And a DNA cassette for transforming Neisseria gonorrhoeae, comprising a promoter for the hydrolase gene, wherein the promoter is operably linked to a transcription control region of the transcription factor. Incubating the transformant of Neisseria gonorrhoeae according to any one of claims 1 to 8, while suppressing a decrease in the amount of conidia formed in the transformant, How to increase. A hydrolase, wherein the transformant of Aspergillus according to any one of claims 1 to 8 is cultured, and the hydrolase is collected from the medium after secreting the protein hydrolase into the medium. Production method.