CN115725632A - Aomsn2 overexpression aspergillus oryzae engineering strain and construction method and application thereof - Google Patents
Aomsn2 overexpression aspergillus oryzae engineering strain and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses an Aomsn2 overexpression aspergillus oryzae engineering strain and a construction method and application thereof, wherein the construction method comprises the following steps: designing a corresponding Aomsn2 primer by taking an Aomsn2 gene in an Aspergillus oryzae sample as a template, and carrying out PCR amplification on an Aspergillus oryzae sample genome by using the Aomsn2 primer to obtain an Aomsn2 cloned gene, wherein the nucleotide sequence of the Aomsn2 cloned gene is shown as SEQ ID NO.1, and the nucleotide sequence of the Aomsn2 primer is shown as SEQ ID NO. 2-3; carrying out enzyme digestion and enzyme linkage treatment on the Aomsn2 cloned gene and an expression vector pEX2B to construct an Aomsn2 overexpression vector; and transforming the Aomsn2 overexpression vector into aspergillus oryzae by adopting a mediated protoplast transformation method to construct and obtain the Aomsn2 overexpression aspergillus oryzae engineering strain. According to the invention, through research, the Aomsn2 gene can improve the yield of kojic acid in aspergillus oryzae by controlling and regulating the transcription factor of a kojic acid gene cluster, so that a foundation is laid for developing an industrial brewing strain with high yield of kojic acid, and a theoretical foundation is provided for utilizing the industrial application of aspergillus oryzae and the production of kojic acid.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to an Aomsn2 overexpression aspergillus oryzae engineering bacterium and a construction method and application thereof.
Background
Aspergillus oryzae (Aspergillus oryzae) is an aerobic fungus that has been identified as a safe producing strain by the FDA and WHO in the United states. Aspergillus oryzae can produce amylase, glucoamylase, cellulase, phytase, etc. in addition to protease. In comparison to E.coli and yeast, aspergillus oryzae has powerful post-translational modification functions, such as glycosylation and protein folding; compared with high-grade organisms such as plants and insects, the aspergillus oryzae grows quickly, has low requirements on the nutritional environment, and is an ideal host for expressing foreign proteins. The aspergillus oryzae exogenous expression system can be used for producing various important organic acids such as malic acid, kojic acid, fatty acid, lactic acid and the like. As such, many researchers are also focusing more and more on the study of Aspergillus oryzae gene expression.
MSN2 encodes a canonical Cys 2 His 2 The gene of the type zinc finger protein is a main transcription factor for controlling the response of the fungal cell to the external adversity stress, has an important role in the response regulation of the fungal cell such as hunger stress, salt stress, oxidation stress, high osmotic pressure stress, low temperature stress and the like, and researches report that the MSN2 gene is related to the number of conidia, colony growth and kojic acid yield in aspergillus nidulans and aspergillus flavus.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide an Aomsn2 overexpression Aspergillus oryzae engineering strain, a construction method and application thereof, and aims to solve the problem that the yield of kojic acid in the secondary metabolite of the existing Aspergillus oryzae is low.
The technical scheme of the invention is as follows:
a construction method of Aomsn2 overexpression Aspergillus oryzae engineering bacteria comprises the following steps:
designing a corresponding Aomsn2 primer by taking an Aomsn2 gene in an Aspergillus oryzae sample as a template, and carrying out PCR amplification on an Aspergillus oryzae sample genome by using the Aomsn2 primer to obtain an Aomsn2 cloned gene, wherein the nucleotide sequence of the Aomsn2 cloned gene is shown as SEQ ID NO.1, and the nucleotide sequence of the Aomsn2 primer is shown as SEQ ID NO. 2-3;
carrying out enzyme digestion and enzyme linkage treatment on the Aomsn2 cloned gene and an expression vector pEX2B to construct an Aomsn2 overexpression vector;
and transforming the Aomsn2 overexpression vector into aspergillus oryzae by adopting a mediated protoplast transformation method to construct and obtain the Aomsn2 overexpression aspergillus oryzae engineering strain.
The construction method of the Aomsn2 overexpression aspergillus oryzae engineering bacteria comprises the step of carrying out a recombination process on an Aomsn2 overexpression vector, wherein a kana resistance marker and an uracil guanosine nutrition marker are arranged on the Aomsn2 overexpression vector.
The invention discloses an Aomsn2 overexpression Aspergillus oryzae engineering bacterium, which is prepared by adopting the construction method of the Aomsn2 overexpression Aspergillus oryzae engineering bacterium.
The application of Aomsn2 over-expression Aspergillus oryzae engineering bacteria is characterized in that the Aomsn2 over-expression Aspergillus oryzae engineering bacteria prepared by the construction method is used for producing kojic acid.
Has the advantages that: the invention constructs Aomsn2 overexpression Aspergillus oryzae engineering bacteria and Aomsn2 knockout engineering strains, and finds that the Aomsn2 gene has larger influence on the growth of Aspergillus oryzae and the synthesis of a secondary metabolite kojic acid through comparative analysis, the Aomsn2 gene can improve the yield of the kojic acid in the Aspergillus oryzae by controlling and regulating transcription factors of kojic acid gene clusters, thereby laying a foundation for developing industrial brewing strains with high yield of the kojic acid, and providing a theoretical foundation for utilizing the industrial application of the Aspergillus oryzae and the production of the kojic acid.
Drawings
FIG. 1 is a flow chart of a construction method of Aomsn2 overexpression Aspergillus oryzae engineering bacteria.
FIG. 2 is a restriction electrophoresis of pPTRII-Cas 9, wherein lanes 1-2 are linearized pPTRII-Cas 9; lane 3 is a control pPTRII-Cas 9.
FIG. 3 is a comparison of the electrophoresis of the Pu6-Aomsn2 fragment, the Pu6-Aomsn2-sgRNA-Tu6 fragment and the Aomsn2-sgRNA-TU6 fragment, wherein lane 1 is the Pu6-Aomsn2 fragment; lane 2,3 is fusion fragment Pu6-Aomsn2-sgRNA-Tu6; lane 4 is an Aomsn2-sgRNA-TU6 fragment control.
FIG. 4 is a graph showing the results of electrophoretic verification of two fragments of Pu6-Aomsn2 and Aomsn2-sgRNA-Tu6, wherein lanes 1-4 are the verification of 4 monoclonal Pu6-Aomsn 2; lanes 5-8 are 4 monoclonal Aomsn2-sgRNA-Tu6 validation.
FIG. 5 is a gel cutting electrophoresis of pEX 2B.
FIG. 6 is a graph showing the result of the Aomsn2 overexpression bacterial suspension verification.
FIG. 7 is a graph showing the screening results of Aomsn2 overexpression positive strains, in which A is a protoplast transformation plate; b is a culture diagram of transformants picked from the protoplast transformation plate.
FIG. 8 is a diagram showing the result of PCR verification of the Aomsn2 overexpression strain, wherein A is an electrophoresis verification diagram, and lane 1 is an Aomsn2 overexpression plasmid control; lanes 2-6 are transformants picked for DNA validation; b is a graph of the result of analysis of the expression pattern of the Aomsn2 overexpression strain.
FIG. 9 is a diagram showing the screening results of transformants of the Aomsn2 knockout strain, wherein A is a protoplast transformation plate; b is a culture diagram of transformants picked from the protoplast transformation plate.
FIG. 10 is a graph showing the results of Aomsn2 sequencing homozygosity.
FIG. 11 is a graph showing the results of analysis of expression patterns of the Aomsn2 knockout strain.
FIG. 12 is a graph showing growth of the Aomsn2 overexpressing strain, wherein A is a graph showing the results of culturing WT and the Aomsn2 overexpressing strain at 30 ℃ for 3 days, and B is a graph showing the analysis of the growth diameter of the strain; c: analyzing the number of the spores of the strain; d: analysis of the strain biomass, (. About.P < 0.05;. About.P < 0.01).
FIG. 13 is a graph showing growth of the Aomsn2 knock-out strain, wherein A is a graph showing the results of culturing WT and the Aomsn2 knock-out strain at 30 ℃ for 3 days, and B is a strain growth diameter analysis; c: analyzing the number of the spores of the strain; d: analysis of the strain biomass, (. About.P < 0.05;. About.P < 0.01).
FIG. 14 is a graph showing the growth of the Aomsn2 overexpressing strain under different treatment conditions, wherein A is a graph showing the results of culturing WT and the Aomsn2 overexpressing strain at 30 ℃ for 3 days under different treatments; b is a graph of the growth diameter analysis of each strain at different treatments, (. About.P < 0.05;. About.P < 0.01).
FIG. 15 is a graph showing the behavior of kojic acid obtained by removing the Aomsn2 gene, wherein A is a graph showing the behavior of each strain when cultured in a solid chromogenic medium at 30 ℃ for 3 days, and red indicates that the strain secretes kojic acid; b is fermentation color development liquid of each strain; c is a kojic acid yield analysis chart of each strain; d is an expression pattern analysis chart of the kojA gene of each strain; e is an analysis graph of expression pattern of kojR gene of each strain, and F is an analysis graph of expression pattern of kojT gene of each strain (. SP < 0.05;. SP < 0.01).
Detailed Description
The invention provides an Aomsn2 overexpression aspergillus oryzae engineering strain and a construction method and application thereof, and the invention is further described in detail below in order to make the purpose, technical scheme and effect of the invention clearer and more clear. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Referring to fig. 1, fig. 1 is a flow chart of a method for constructing an aspergillus oryzae engineered bacterium overexpressed by Aomsn2 according to the present invention, as shown in the figure, the method includes the steps of:
s10, designing a corresponding Aomsn2 primer by taking an Aomsn2 gene in an Aspergillus oryzae sample as a template, and carrying out PCR amplification on an Aspergillus oryzae sample genome by using the Aomsn2 primer to obtain an Aomsn2 cloned gene, wherein the nucleotide sequence of the Aomsn2 cloned gene is shown as SEQ ID NO.1, and the nucleotide sequence of the Aomsn2 primer is shown as SEQ ID NO. 2-3;
s20, carrying out enzyme digestion and enzyme linkage treatment on the Aomsn2 cloned gene and an expression vector pEX2B to construct an Aomsn2 overexpression vector;
s30, transforming the Aomsn2 overexpression vector into Aspergillus oryzae by adopting a mediated protoplast transformation method, and constructing and preparing the Aomsn2 overexpression Aspergillus oryzae engineering strain.
Specifically, the invention constructs the Aomsn2 overexpression Aspergillus oryzae engineering strain and the Aomsn2 knockout engineering strain, and finds that the Aomsn2 gene has great influence on the growth of Aspergillus oryzae and the synthesis of kojic acid as a secondary metabolite of the Aspergillus oryzae through comparative analysis, the Aomsn2 gene can improve the yield of the kojic acid in the Aspergillus oryzae by controlling and regulating transcription factors of a kojic acid gene cluster, so that a foundation is laid for developing an industrial brewing strain with high yield of the kojic acid, and a theoretical foundation is provided for the application of the Aspergillus oryzae in industry and the production of the kojic acid.
In some embodiments, the invention also provides an Aomsn2 overexpression Aspergillus oryzae engineering bacterium, which is prepared by the method for constructing the Aomsn2 overexpression Aspergillus oryzae engineering bacterium.
In some embodiments, the invention further provides application of the Aomsn2 overexpression Aspergillus oryzae engineering bacteria, wherein the Aomsn2 overexpression Aspergillus oryzae engineering bacteria prepared by the construction method are used for producing kojic acid.
The invention is further illustrated by the following specific examples:
example 1
Construction of the Aomsn2 knockout vector:
based on the CRISPR/Cas9 system, the CRISPR/Cas9 system comprises two components: one part is Cas9 endonuclease and the other part is single stranded guide RNA (sgRNA). Wherein, the Cas9 endonuclease is an RNA-guided DNA lyase, the Cas9 endonuclease is controlled by a gpdA promoter, and the DNA which can be targeted and recognized by the Cas9 endonuclease is determined by a guide RNA sequence with the length of 20 bp; sgRNA can function as a guide, directing the Cas9 enzyme to recognize and cleave the target sequence upstream of the protospacer motif (PAM). In the embodiment, the Aomsn2 knockout vector uses pPTR II as a parent vector, and a vector pPTR II-Cas 9 is obtained through vector transformation, wherein the vector pPTR II-Cas 9 is used for constructing the Aomsn2 knockout vector, contains a resistance screening marker of pyrithione, and is used for enzyme digestion of the vector pPTR II-Cas 9 by SmaI enzyme, and agarose gel is used for detecting whether the vector is cut, and the result is shown in FIG. 2.
Firstly, using Aspergillus oryzae 3.042 strain genome DNA as a template, and utilizing a primer containing a target gene sequence of Aomsn2 bp to amplify a U6 promoter to obtain a Pu6-Aomsn2 sequence, wherein the used primer is shown in Table 1, and the corresponding nucleotide sequence is shown in SEQ ID NO. 4-7.
TABLE 1 primer sequences
| PU6-Aomsn2-R | CACTCCAGGACTGGCGGGGTACTTGTTCTTCTTTACAATGATTTATTTA |
| TU6-Aomsn2-F | ACCCCGCCAGTCCTGGAGTGGTTTTAGAGCTAGAAATAGCAAGTTAAA |
| PU6-F | CGACTCTAGAGGATCCCCGGGTAATGCCGGCTCATTCAAA |
| TU6-R | AATTCGAGCTCGGTACCCGGGAGCAGCTCTATATCACGTGACG |
The U6 terminator sequence and sgRNA sequence were synthesized by the company (i.e., sgRNA-Tu 6). And (3) amplifying and connecting the targeting sequence of the Aomsn2 to the sgRNA-Tu6 by using the sgRNA-Tu6 as a template to obtain the sequence of the Aomsn2-sgRNA-Tu 6. The two fragments of Pu6-Aomsn2 and Aomsn2-sgRNA-Tu6 were fused to obtain a fused fragment Pu6-Aomsn2-sgRNA-Tu6, as shown in FIG. 3. The fusion fragment Pu6-Aomsn2-sgRNA-Tu6 is recombined and connected to a linearized pPTRII-Cas 9 vector and is transformed by an Escherichia coli host. Selecting monoclonal shake bacteria, carrying out PCR verification on the bacteria liquid, respectively verifying two fragments of Pu6-Aomsn2 and Aomsn2-sgRNA-Tu6 by the PCR of the bacteria liquid so as to ensure that the two ends are fused together and successfully transformed, and naming the constructed recombinant plasmid as pPTR II-Cas 9-Aomsn2, wherein the result is shown in figure 4. The plasmid pPTRII-Cas 9-Aomsn2 is transformed into aspergillus oryzae by a PEG (polyethylene glycol) mediated protoplast transformation method, and the Aomsn2 knockout engineering strain is constructed.
Example 2
Construction of Aomsn2 over-expressed Aspergillus oryzae engineering bacteria:
the pEX2B vector is used for constructing an Aomsn2 gene overexpression vector and has a kana resistance marker and a uracil guanosine nutrition marker. The vector pEX2B was digested with both fast-cutting enzyme AflII and BamHI, and the DsRed fragment was excised, and the vector was examined for cleavage using agarose gel, the results of which are shown in FIG. 5. The genomic DNA of Aspergillus oryzae 3.042 is used as a template, an Aomsn2 fragment is amplified through a designed Aomsn2 primer, a linearized pEX2B vector and the Aomsn2 fragment are cut into gel, recovered, transformed into Trans10 after recombinant connection, monoclonal shake bacteria are picked, and bacteria liquid PCR verification is carried out, and the result is shown in figure 6. And (3) sequencing the positive strains by a sequencing company, carrying out medium-quality-improvement on the sequenced positive strains, and transforming the plasmids (the Aomsn2 overexpression vector) into the aspergillus oryzae by adopting a PEG-mediated protoplast transformation method to construct and prepare the Aomsn2 overexpression aspergillus oryzae engineering bacteria.
Example 3
After 3 to 4 days of culturing the Aomsn 2-overexpressing engineered Aspergillus oryzae strain constructed in example 2, transformants grown from the middle of the medium were picked and transferred to a CD medium, as shown in FIG. 7. After culturing for 2-3 days, the outermost hyphae were picked with a medium-sized tip, 50uLTE medium-boiled DNA was added as a template, PCR was performed with primers on the vector, and Aomsn2 was amplified using pEX2B-Aomsn2 plasmid as a template for control, and agarose gel electrophoresis was used to detect whether the vector was transferred into Aspergillus oryzae, the results being shown in FIG. 8. Two over-expressed strains were obtained and named OE-Aomsn2-1 and OE-Aomsn2-2, respectively. And two over-expression strains and WT RNA are extracted, and the expression level of the Aomsn2 is analyzed by using RT-PCR, and the result shows that: the expression level of the Aomsn2 gene in two Aomsn2 overexpression strains is obviously higher than that of the wild strain. Two over-expression strains are spread on KA culture medium for amplification culture, spores are collected, glycerol is added for bacterium preservation (bacterium liquid: 50% glycerol = 1:1), and the mixture is stored in a refrigerator at the temperature of minus 80 ℃ for later use.
The Aomsn2 knock-out engineered strain constructed in example 1 was cultured, and after the transformants grew out, the transformants were transferred to a CD + PT medium containing 0.1ug/mL thiamine pyridine, as shown in FIG. 9. After culturing for 2-3 days, picking the outer most circle of hyphae by using a medium-sized gun head, adding 50uLTE medium-boiled DNA as a template, amplifying an Aomsn2 fragment by using a primer containing a targeting sequence for verification, detecting by using agarose gel electrophoresis, and cutting and sequencing the correct target band. The sequencing results are shown in FIG. 10, wherein one knockout strain has a base A inserted into the exon, resulting in the following base frame shift mutation, and is named as delta Aomsn2-1, and the other strain has a deletion of 20bp on the exon, resulting in the premature translation termination, and is named as delta Aomsn2-2, and the sequencing results are all homozygous. In FIG. 11, the expression level of the Aomsn2 gene was significantly down-regulated in both the Δ Aomsn2-1 and Δ Aomsn2-2 knock-out strains compared to wild type. The delta Aomsn2-1 and delta Aomsn2-2 knockout strains are coated with a KA culture medium for expanding culture and spore collection, and glycerol is added for bacterium preservation (bacterium liquid: 50% glycerol = 1:1) and stored in a refrigerator at-80 ℃ for later use.
Example 4
Phenotypic analysis was performed on two Aomsn2 overexpressing engineering bacteria and two Aomsn2 knockout strains:
preparing a CD solid culture medium, respectively coating spores of the Aomsn2 overexpression Aspergillus oryzae engineering bacteria and the Aomsn2 knockout engineering strain in example 3 stored at-80 ℃ on a KA culture medium for activation, collecting the spores by using 0.9-percent NaCl solution, storing the collected spore solution in a refrigerator at 4 ℃ for 10-15 days, taking 3uL of the same amount of spore solution for spotting, and culturing in the incubator at 30 ℃ for 3 days. The medium was treated differently with 120ug/mL Congo Red, 60uM K3, 1M NaCl, respectively. In the application process of aspergillus oryzae, the living environment becomes worse due to the self-existence and metabolism, for example, under the oxidation condition, the oxidation damage is easy to occur, and in order to investigate the function of the Aomsn2, congo red and vitamin K3 oxidant are used to create the oxidation condition. Aspergillus oryzae is often used in industry to ferment food, so it is also very necessary to explore a physiological condition of Aspergillus oryzae under NaCl salt stress.
1. Measurement of diameter and number of spores of each strain:
the spore solution spotted CD solid medium was cultured in a 30 ℃ incubator for 3 days, and then the growth diameter was measured (cross method), and the data was recorded.
It is rinsed with 1500. Mu.L of water (0.25% triton100 added), vortexed vigorously using a vortexer to thoroughly break up the sporangia, and the spore count is calculated using a hemocytometer, and if the concentration is too high, diluted appropriately.
2. Determination of the dry weight of each strain:
100uL of the bacterial liquid is smeared on a CD culture medium paved with glass paper and cultured for 3 days at 30 ℃. Scraping, drying and weighing.
3. Measurement of growth diameter under different treatments:
the above-mentioned strains treated differently were incubated at 30 ℃ for 3 days in an incubator, and then the growth diameter was measured (cross method) and data was recorded.
The above test results are shown in FIGS. 12 to 13, and it can be seen from the results of FIGS. 12 to 13 that overexpression of the Aomsn2 gene reduces the growth diameter and biomass of the strain, increasing the number of spores; and knocking out the Aomsn2 gene can increase the growth diameter and biomass of the strain and reduce the number of spores. Thus, the Aomsn2 gene has an important effect on the growth of Aspergillus oryzae.
4. The influence of the Aomsn2 gene on the growth of aspergillus oryzae under stress is explored:
respectively using Congo red and microorganism K 3 And respectively treating the engineering strains by using sodium chloride. The results are shown in FIG. 14, using the same procedure as above. The results show that the knockout of the Aomsn2 gene has no obvious influence on the growth of Aspergillus oryzae in a stress environment, and the overexpression of the Aomsn2 gene makes Aspergillus oryzae more sensitive to external stress, which indicates that the Aomsn2 gene is a response factor for the growth of Aspergillus oryzae under stress.
5. The influence of the Aomsn2 gene on the Aspergillus oryzae secondary metabolite kojic acid was explored, and the content of kojic acid was determined using the method reported by Bentley:
taking 3 mu L of the spot plate of each engineering strain, putting the spot plate on a CD kojic acid solid culture medium and a 3mmol/L FeCl3 plate, culturing for 3 days at 30 ℃, and observing.
Inoculating 100uL of the engineering strains into 40mL of fermentation liquor of an LCD kojic acid culture medium (the fermentation bottle is a 250mL conical flask), and fermenting for 7 days at the temperature of 30 ℃ and 200rpm of a shaking table.
Centrifuging 2mL of fermentation broth, and dissolving 10g of FeCl in the supernatant according to the ratio of color-developing agent (1% of FeCl3, ddH2O) 3 ·6H 2 O, adding 22.5mL concentrated hydrochloric acid, and fixing the volume to 1L) =1:2, preparing the solution to be detected, generally taking 500uL of supernatant and 1000uL of color developing solution in a clean 2.0mL centrifuge tube, and then adding 500uL ddH2O. The absorbance at 500nm was measured. The control group was prepared with 1mL ddH2O and 1mL color developer (1% FeCl3). And substituting the obtained data into a kojic acid standard curve to calculate the amount of kojic acid produced by each strain. It was found that knocking out the Aomsn2 gene inhibits Aspergillus oryzae kojic acid synthesis. WT and AomThe sn2 knockout strain is fermented in a liquid kojic acid CD culture medium for 3 days, aspergillus oryzae hyphae are collected, RNA is extracted, and RT-PCR analysis is carried out, so that knockout of Aomsn2 inhibits the expression of kojA and kojT genes related to kojic acid synthesis, and the kojR is not influenced, and the result is shown in figure 15.
It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.
Claims (4)
1. A construction method of Aomsn2 over-expression Aspergillus oryzae engineering bacteria is characterized by comprising the following steps:
designing a corresponding Aomsn2 primer by taking an Aomsn2 gene in an Aspergillus oryzae sample as a template, and carrying out PCR amplification on an Aspergillus oryzae sample genome by using the Aomsn2 primer to obtain an Aomsn2 cloned gene, wherein the nucleotide sequence of the Aomsn2 cloned gene is shown as SEQ ID NO.1, and the nucleotide sequence of the Aomsn2 primer is shown as SEQ ID NO. 2-3;
carrying out enzyme digestion and enzyme linkage treatment on the Aomsn2 cloned gene and an expression vector pEX2B to construct an Aomsn2 overexpression vector;
and transforming the Aomsn2 overexpression vector into aspergillus oryzae by adopting a mediated protoplast transformation method to construct and obtain the Aomsn2 overexpression aspergillus oryzae engineering strain.
2. The method for constructing Aomsn 2-overexpressed Aspergillus oryzae engineering bacteria according to claim 1, wherein the Aomsn2 overexpression vector is provided with a kanamycin resistance marker and a uracil guanosine nutrition marker.
3. An Aomsn2 over-expressed Aspergillus oryzae engineering bacterium, which is prepared by the method for constructing the Aomsn2 over-expressed Aspergillus oryzae engineering bacterium according to any one of claims 1 to 2.
4. Use of an engineered aspergillus oryzae with an overexpression of Aomsn2 in the production of kojic acid, wherein the engineered aspergillus oryzae with the overexpression of Aomsn2 prepared by the construction method of any one of claims 1-2 is used.
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