Application of Aokap12 gene and encoding protein thereof in improving yield of kojic acid of aspergillus oryzae

Abstract

The invention belongs to the technical field of microbial genetic engineering, and particularly relates to an application of an Aokap12 gene and an encoding protein thereof in improving the kojic acid yield of aspergillus oryzae. According to the invention, nucleotide sequences from 64th site to 83rd site at the downstream of an Aspergillus oryzae Aokap12 gene translation code starting site are taken as target sequences, a CRISPR / Cas9 technology is utilized to construct a recombinant vector which destroys an open reading frame of the Aokap12 gene, efficient fixed-point knockout of the Aokap12 is realized through a gene editing technology, and the construction efficiency of an engineering strain for high-yield kojic acid based on Aokap12 gene mutation is effectively improved; through gene editing of the Aokap12 gene, the kojic acid yield of Aspergillus oryzae is significantly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial genetic engineering, and particularly relates to application of an Aokap12 gene and a coded protein thereof in improving the kojic acid yield of Aspergillus oryzae. Background Art

[0002] Aspergillus oryzae is an industrial filamentous strain with important application value. Aspergillus oryzae has the characteristics of loose growth conditions, rapid growth, large spore production and rich enzyme system, and is widely used in the food field. Moreover, Aspergillus oryzae does not produce toxins and is a food-grade generally recognized as safe (GRAS) strain certified by the World Health Organization and the United States. Because Aspergillus oryzae has a strong protein secretion ability, including protease, amylase, saccharifying enzyme, lipase, etc., Aspergillus oryzae is an excellent strain for producing enzyme preparations. In addition, the metabolites of Aspergillus oryzae are also widely used in industries such as cosmetics, medicine, and agriculture, such as kojic acid.

[0003] The chemical name of kojic acid is 2-hydroxymethyl-5-hydroxy-4-pyrone, which is a secondary metabolite mainly produced by aerobic fermentation of Aspergillus oryzae. Kojic acid has antibacterial, antioxidant and tyrosinase inhibitory properties, so it is widely used in food, medicine, cosmetics and other industries. According to statistics, the kojic acid market size in 2020 was US$35 million, and China is the main producer of kojic acid. Therefore, achieving efficient biosynthesis of kojic acid has become a research focus.

[0004] At present, the production of kojic acid mainly relies on Aspergillus oryzae to ferment glucose as raw material. At present, the efficient production of kojic acid by Aspergillus oryzae mainly starts from the screening of Aspergillus oryzae strains with high kojic acid production and the optimization of fermentation conditions. The selection of excellent strains with high kojic acid production is mainly achieved through mutagenesis screening, but mutagenesis breeding has some disadvantages, such as uncertain mutation direction, more harm than good, long breeding cycle, and heavy workload. With the development of synthetic biology, the use of synthetic biology technology to transform Aspergillus oryzae in a targeted manner and break through the limitations of natural evolution, and the construction of kojic acid efficient production strains will have broader application prospects. However, the premise is the extensive mining of gene targets that increase the yield of kojic acid in Aspergillus oryzae. Summary of the invention

[0005] The invention provides application of an Aokap12 gene and a protein encoded therein in improving the kojic acid yield of Aspergillus oryzae, which can be used to improve the kojic acid yield of Aspergillus oryzae.

[0006] One of the purposes of the present invention is to provide a gene for improving the kojic acid production of Aspergillus oryzae, wherein the gene is the Aspergillus oryzae Aokap12 gene; the Aspergillus oryzae Aokap12 gene has any one of the following nucleotide sequences:

[0007] 1) The nucleotide sequence shown in SEQ ID NO.1;

[0008] 2) A nucleotide sequence having more than 90% homology with the nucleotide sequence shown in SEQ ID NO.1 and encoding the amino acid sequence shown in SEQ ID NO.2.

[0009] The second object of the present invention is to provide the application of the above gene in improving the kojic acid production of Aspergillus oryzae.

[0010] Furthermore, the application is specifically embodied as: knocking out the Aokap12 gene in Aspergillus oryzae to construct a positive engineering bacterium with the Aokap12 gene knocked out, thereby improving the kojic acid production of Aspergillus oryzae.

[0011] Furthermore, knocking out the Aokap12 gene can increase the kojic acid production of Aspergillus oryzae by 34%.

[0012] The third object of the present invention is to provide an editing recombinant vector for editing the Aokap12 gene of Aspergillus oryzae. The editing recombinant vector of the Aokap12 gene of Aspergillus oryzae is a recombinant vector constructed by using the CRISPR / Cas9 technology and capable of disrupting the open reading frame of the Aokap12 gene; the editing recombinant vector of the Aokap12 gene of Aspergillus oryzae contains the target sequence of the Aokap12 gene of Aspergillus oryzae.

[0013] Among them, the target sequence of the Aokap12 gene of Aspergillus oryzae is shown in SEQ ID NO.3.

[0014] The fourth object of the present invention is to provide a method for improving the kojic acid production of Aspergillus oryzae, including the following steps: transferring the above-mentioned editing recombinant vector of the Aokap12 gene of Aspergillus oryzae into the protoplast of Aspergillus oryzae, knocking out the Aokap12 gene in Aspergillus oryzae, and identifying the transformant to identify the gene editing situation of the Aokap12 gene, obtaining a positive engineering bacterium with the Aokap12 gene knocked out, thereby improving the kojic acid production of Aspergillus oryzae.

[0015] Furthermore, the method for improving the kojic acid production of Aspergillus oryzae specifically includes the following steps:

[0016] 1) Using the vector pPTRII-Cas9-kojA as a template, carrying the Aspergillus oryzae U6 promoter PU6, sgRNA and the Aspergillus oryzae U6 terminator TU6 sequence, amplifying the Aspergillus oryzae U6 promoter PU6 with the primer PU6-F and the primer PU6-Aokap12-R to obtain the PU6-Aokap12 target fragment, and amplifying the sgRNA and the Aspergillus oryzae U6 terminator TU6 with the primer TU6-Aokap12-F and the primer TU6-R to obtain the sgRNA-Aokap12 target fragment;

[0017] 2) Using the PU6-Aokap12 target fragment and the sgRNA-Aokap12 target fragment as templates, perform overlapping PCR amplification and purification with primer PU6-F and primer TU6-R to obtain an sgRNA expression cassette containing the Aokap12 gene target sequence;

[0018] 3) Digest the pPTRII-Cas9 vector and purify and recover to obtain a linearized pPTRII-Cas9 vector. Recombinantly ligate the linearized pPTRII-Cas9 vector with the PCR amplification product of the sgRNA expression cassette fragment purified and recovered in step 2) to obtain an Aspergillus oryzae Aokap12 gene editing recombinant vector, namely the Aspergillus oryzae Aokap12 gene CRISPR / Cas9 vector pPTRII-Cas9-Aokap12 containing the Aokap12 gene target sequence;

[0019] 4) Transfer the Aspergillus oryzae Aokap12 gene editing recombinant vector into Aspergillus oryzae protoplasts, knockout the Aokap12 gene in Aspergillus oryzae and conduct identification to obtain a positive engineering strain with the Aokap12 gene knocked out.

[0020] Furthermore, the sequences of the primers used for amplifying the knockout target when constructing the Aspergillus oryzae Aokap12 gene editing recombinant vector are as follows:

[0021] Primer PU6-F: CGACTCTAGAGGATCCCCGGGTAATGCCGGCTCATTCAAA (SEQ ID NO.4);

[0022] Primer PU6-Aokap12-R: GAAGGTCCTGCGCCCACCGAACTTGTTCTTCTTTACAATGATTTATTTA (SEQ ID NO.5);

[0023] Primer TU6-Aokap12-F: TCGGTGGGCGCAGGACCTTCGTTTTAGAGCTAGAAATAGCAAGTTAAA (SEQ ID NO.6);

[0024] Primer TU6-R: AATTCGAGCTCGGTACCCGGGAGCAGCTCTATATCACGTGACG (SEQ ID NO.7).

[0025] Furthermore, the sequences of the primers used for identifying the gene editing of the positive engineering strain with the Aokap12 gene knocked out are as follows:

[0026] Primer CRISPR-S-Aokap12-F: GACTCCGTCGCGTATCGTCG (SEQ ID NO.8);

[0027] Primer CRISPR-S-Aokap12-R: AGCCACCGGGGATTCCAGTA (SEQ ID NO.9).

[0028] The beneficial effects of the present invention are as follows:

[0029] (1) The present invention provides a new gene for improving kojic acid production in Aspergillus oryzae: the Aokap12 gene. By gene editing the Aokap12 gene, the kojic acid production in Aspergillus oryzae can be significantly increased. The present invention provides an important functional gene for the genetic improvement of kojic acid production in Aspergillus oryzae.

[0030] (2) The present invention realizes the efficient site-directed knockout of Aokap12 through gene editing technology. The present invention discovers that the nucleotide sequence from the 64th to the 83rd position downstream of the translation start codon of the Aokap12 gene in Aspergillus oryzae can be used as a target sequence, which can efficiently achieve the site-directed knockout of the Aokap12 gene and effectively improve the construction efficiency of high-yield kojic acid engineering strains based on Aokap12 gene mutation. Description of the Drawings

[0031] Figure 1 It is a schematic diagram of the mutation type of the nucleotide sequence of the Aokap12 gene in the homozygous mutant strain of the Aokap12 gene under the background of wild-type Aspergillus oryzae 3.042.

[0032] Figure 2 It is a schematic diagram of the mutation type of the amino acid sequence of the Aokap12 gene in the homozygous mutant strain of the Aokap12 gene under the background of wild-type Aspergillus oryzae 3.042.

[0033] Figure 3 It is a result diagram of kojic acid production analysis of Aspergillus oryzae 3.042 and Aokap12 mutant bacteria. Detailed Embodiments

[0034] The present invention will be further described in detail below in combination with specific embodiments. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0035] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0036] The Aspergillus oryzae strain in the following examples: Aspergillus oryzae 3.042 (CICC 40092), which can be obtained by the public from the China Center for Industrial Culture Collection of Microorganisms.

[0037] In the examples of the present invention, the CRISPR-Cas9 vector is the pPTRII-Cas9 vector, which has been described in Yuzhen Li # , Huanxin Zhang # , Junxia Fan # , Ziming Chen, Tianming Chen, Bin Zeng, Zhe Zhang *A highly efficient identification of mutants generated by CRISPR / Cas9 using the non-functional DsRed assisted selection in Aspergillus oryzae, World Journal of Microbiology and Biotechnology, 2021, 37:132, which can be obtained by the public from the College of Life Sciences, Jiangxi Science and Technology Normal University. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0038] Example 1: Design of specific sgRNA targeting the Aokap12 gene of Aspergillus oryzae

[0039] Taking the CDS sequence of the Aokap12 gene of Aspergillus oryzae RIB40 as a reference, a specific sgRNA targeting the Aokap12 gene was designed using the CRISPRdirect web server (https: / / crispr.dbcls.jp / ). The nucleotide sequence (CDS sequence) of the Aokap12 gene is shown in SEQ ID NO.1, and the amino acid sequence of the Aokap12 gene is shown in SEQ ID NO.2.

[0040] SEQ ID NO.1:

[0041] ATGCAACTCACTCGTTTATCTGCCGTCCTTCTTCTCTCCGGAGCCTACGCTGCAGTAGCACCGGAAGGTCCTGCGCCCACCGAGATCCCGGGTGGTTGCAACCCAGCTCATCCTGGAAGCTGTCCCGAGACTGCCACTGAGATTCCCGGAGGTCTGGGCTGCAATCCTGCTCACCCTGGCAGCTGCCCTACCCCAACTGATATTCCCGGTGGATGTAACCCAGCGCATCCCGGCAGCTGCCCTACTCCTACCGAGATCCCTGGAGGCTGCAACCCAGCTCACCCTGGAAGCTGCCCCGAATCCACCGAAATCCCCGGTGGTCTGGGCTGCAACCCAGCTCATCCTGGCAGCTGCCCTGAGTCCACCCAAATCCCTGGTGGATGTAACACTGCTCACCCAGGTAGCTGCCCTACGCCAACCGAGATCCACGGAGGTTGCAACCCAGCGCATCCCGGCAGCTGTCCTACTCCTACTGGAATCCCCGGTGGCTGCAACCCCGCTCACCCCGGAAGCTGCCCCAGCGTGACCGTGGTGTCCGGTAGCTCGACCTACCTCTCCCCCGTCGCCGCCGTGACCCCATCTGGCTTCAGCAGCATCGCTGGTATTCCTGGTGGCTGCAACCCAGCTCACCCTGGAAGCTGCCCCAGCGTGACTGTTGTGTCTGGCAGCTCCACCTACATGTCCCCTGTTCCTGTTGCTACTTCCTCTGGCAAGTTCTCGGGTAATCTGACTTCCTCTAGCCTTAATCCTCTTTTCACCGGTGCTGCCCCGAACAACCAGCTCCACTACGGCGCTCTCCTTACCGGTGTCATTGCTCTTGCTGCCCGTGCTTTCTTCTAA

[0042] SEQ ID NO.2:

[0043] MQLTRLSAVLLLSGAYAAVAPEGPAPTEIPGGCNPAHPGSCPETATEIPGGLGCNPAHPGSCPTPTDIPGGCNPAHPGSCPTPTEIPGGCNPAHPGSCPESTEIPGGLGCNPAHPGSCPESTQIPGGCNTAHPGSCPTPTEIHGGCNPAHPGSCPTPTGIPGGCNPAHPGSCPSVTVVSGSSTYLSPVAAVTPSGFSSIAGIPGGCNPAHPGSCPSVTVVSGSSTYMSPVPVATSSGKFSGNLTSSSLNPLFTGAAPNNQLHYGALLTGVIALAARAFF

[0044] Among them, the nucleotide sequence from the 64th to the 83rd position downstream of the translation start codon of the Aspergillus oryzae Aokap12 gene is used as the target sequence; the specific sgRNA targeting the Aokap12 gene contains the target sequence of the Aspergillus oryzae Aokap12 gene, and the nucleotide sequence of the target sequence is: GAAGGTCCTGCGCCCACCGA (SEQ ID NO.3), and the corresponding PAM sequence is CGG.

[0045] Example 2: Construction of an sgRNA expression cassette containing the Aokap12 gene target sequence

[0046] Using the vector pPTRII-Cas9-kojA constructed by the previous applicant as a template (this vector has been described in YuzhenLi # , Huanxin Zhang # , Junxia Fan #, Ziming Chen, Tianming Chen, Bin Zeng, Zhe Zhang *A highly efficient identification of mutants generated by CRISPR / Cas9 using the non-functional DsRed assisted selection in Aspergillus oryzae*, World Journal of Microbiology and Biotechnology, 2021, 37:132). The vector pPTRII-Cas9-kojA contains the Aspergillus oryzae U6 promoter PU6, the kojA gene targeting sequence, the sgRNA, and the Aspergillus oryzae U6 terminator TU6 sequence. The Aspergillus oryzae U6 promoter PU6 was amplified by PCR using the primer PU6-F and the primer PU6-Aokap12-R to obtain the PU6-Aokap12 target fragment containing the Aspergillus oryzae U6 promoter PU6 and the Aokap12 gene target sequence. The sgRNA and the Aspergillus oryzae U6 terminator TU6 were amplified by PCR using the primer TU6-Aokap12-F and the primer TU6-R. During the amplification process, the target sequence of the Aokap12 gene was ligated in front of the sgRNA and the Aspergillus oryzae U6 terminator TU6 through a primer linker to form the Aokap12 gene target sequence-sgRNA-TU6 fragment, thereby obtaining the sgRNA-Aokap12 target fragment containing the specific sgRNA targeting the Aokap12 gene and the Aspergillus oryzae U6 terminator TU6. Phanta Super-Fidelity DNA polymerase (Novoprotein, China) was used for amplification. The PCR amplification system was as follows: 1 μL of template DNA (pPTRII-Cas9-kojA plasmid), 4 μL of primers (10 mM) (i.e., 2 μL each of primer PU6-F and primer PU6-Aokap12-R), 1 μL of dNTP Mix (10 mM), 10 μL of 5×SF Buffer, 1 μL of Phanta Super-Fidelity DNA polymerase, and 33 μL of ddH2O. The PCR amplification reaction program was: pre-denaturation at 95 °C for 3 min, denaturation at 95 °C for 10 sec, annealing at 55 °C for 30 sec, extension at 72 °C for 1 min, 35 cycles, and final extension at 72 °C for 5 min. Electrophoresis detection showed that the PCR product PU6-Aokap12 target fragment was about 550 bp, and the sgRNA-Aokap12 target fragment was about 200 bp.

[0047] Take 1 μL each of the above-mentioned PU6-Aokap12 target fragment and sgRNA-Aokap12 target fragment as templates, and use primer PU6-F and primer TU6-R to perform PCR amplification according to the above PCR amplification system and PCR amplification reaction procedure for 35 cycles. The product size is about 750 bp. The product recovered by gel cutting is the sgRNA expression cassette containing the Aokap12 gene target sequence.

[0048] Among them, the nucleotide sequence of primer PU6-F is:

[0049] CGACTCTAGAGGATCCCCGGGTAATGCCGGCTCATTCAAA(SEQ ID NO.4);

[0050] The nucleotide sequence of primer PU6-Aokap12-R is:

[0051] GAAGGTCCTGCGCCCACCGAACTTGTTCTTCTTTACAATGATTTATTTA(SEQ ID NO.5);

[0052] The nucleotide sequence of primer TU6-Aokap12-F is:

[0053] TCGGTGGGCGCAGGACCTTCGTTTTAGAGCTAGAAATAGCAAGTTAAA(SEQ ID NO.6)

[0054] The nucleotide sequence of primer TU6-R is:

[0055] AATTCGAGCTCGGTACCCGGGAGCAGCTCTATATCACGTGACG(SEQ ID NO.7).

[0056] Example 3: Construction of Aspergillus oryzae Aokap12 gene CRISPR / Cas9 vector

[0057] Digest the pPTRII-Cas9 vector with the restriction endonuclease Sma I (TaKaRa) (37 °C, 1 h). The digestion system is as follows: 2 μg of pPTRII-Cas9 vector, 5 μL of 10× QuickCut Green Buffer, 2 μL of Sma I, and ddH2O is added to make up to 50 μL. The digested product is directly purified using a PCR product recovery kit to obtain the linearized pPTRII-Cas9 vector. Then, the linearized pPTRII-Cas9 vector and the PCR product of the above-mentioned sgRNA expression cassette fragment containing the Aokap12 gene target sequence are recombinantly ligated using the ClonExpress rapid cloning recombinase (Vazyme, China). The recombinant ligation system is as follows: linearized pPTRII-Cas9 vector: 200 ng; PCR product of the sgRNA expression cassette fragment containing the Aokap12 gene target sequence: 80 ng; 5× Buffer: 2 μL; recombinase Exnase II (Vazyme product number: C112-02): 2 μL; ddH2O is added to make up the total volume to 10 μL. Incubate at 37 °C for 30 min, and after the reaction, cool to 4 °C or immediately place on ice to obtain the ligation product.

[0058] Take 2 μL of the ligation product and transform Escherichia coli DH5α (TransGen Biotech, China) by heat shock method. Coat it on an ampicillin-resistant LB plate to screen for positive clones. Pick a single clone and shake the bacteria at 37 °C for 12 hours, and finally perform a bacterial liquid PCR amplification reaction. The primers used for the PCR amplification reaction are primer PU6-F (SEQ ID NO.4) and primer TU6-R (SEQ ID NO.7). The electrophoresis detection result shows that the band at 750 bp is the target band, and the Aspergillus oryzae Aokap12 gene CRISPR / Cas9 vector pPTRII-Cas9-Aokap12 containing the Aokap12 gene target sequence is obtained.

[0059] Example 4: Genetic transformation of Aspergillus oryzae

[0060] 1) Transfer 100 μL of freshly harvested Aspergillus oryzae 3.042 spore solution to 100 mL of DPY liquid medium (2% glucose, 1% peptone, 0.5% yeast extract, 0.5% potassium dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate, pH 5.5), and culture at 200 rpm and 30 °C for 16 - 20 h.

[0061] 2) Filter and collect the mycelia with double-layer lens paper, and lyse the above-mentioned cultured Aspergillus oryzae mycelia with 1% Yatalase (TaKaRa) and 1.5% lyticase (Sigma), and incubate at 60 rpm and 30 °C for 3 h to prepare Aspergillus oryzae protoplasts.

[0062] 3) Mix 10 μg of the Aspergillus oryzae Aokap12 gene CRISPR / Cas9 vector pPTRII-Cas9-Aokap12 with 200 μL of Aspergillus oryzae protoplasts and incubate on ice for 30 min.

[0063] 4) Add 250 μL, 250 μL, and 850 μL of PEG buffer (60% PEG 4000, 50 mM calcium chloride dihydrate, 10 mM Tris-HCl, pH 7.5) sequentially in three times and gently mix.

[0064] 5) After standing at room temperature in the dark for 20 min, dilute the above mixed protoplasts with 5 mL of washing buffer (1.2 M sorbitol, 50 mM calcium chloride dihydrate, 35 mM sodium chloride, 10 mM Tris-HCl, pH 7.5) and centrifuge at 4°C for 8 min to collect the protoplasts.

[0065] 6) Then add 5 mL of M+Met medium containing 0.5% agar (0.2% ammonium chloride, 0.1% ammonium sulfate, 0.05% potassium chloride, 0.05% sodium chloride, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate, 0.002% ferrous sulfate heptahydrate, 2% glucose, 0.15% methionine, 1.2 M sorbitol, pH 5.5), mix well, pour it onto a plate of M+Met medium containing 1.5% agar, and culture at 30°C for 3 - 5 days.

[0066] 7) After the mycelia grow, transfer the mycelia to CD medium containing 0.1 μg / ml pyrithiamine (2% glucose, 0.2% sodium nitrate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.05% potassium chloride, 0.05% sodium chloride, 0.002% ferrous sulfate, pH 5.5) and culture at 30°C for 3 - 5 days to screen for positive bacteria using pyrithiamine.

[0067] Example 5: Identification of Aspergillus oryzae Aokap12 gene-edited mutants

[0068] Pick the mycelia grown on the above CD medium containing 0.1 μg / ml pyrithiamine, put them into a 200 μL centrifuge tube, add 100 μL of 25 mM NaOH solution, and heat at 100 °C for 20 min. Using KOD DNA polymerase (TOYOBO), use the supernatant as a template, and use primer CRISPR-S-Aokap12-F (SEQ ID NO.8) and primer CRISPR-S-Aokap12-R (SEQ ID NO.9) to PCR amplify the target region sequence of the Aokap12 gene. The PCR amplification system is: 2 μL of supernatant DNA template, 4 μL of primers (i.e., 2 μL each of primer CRISPR-S-Aokap12-F and primer CRISPR-S-Aokap12-R), 4 μL of dNTPs, 25 μL of 2× Buffer, 1 μL of KOD DNA polymerase, 14 μL of ddH2O. The PCR amplification reaction program is: pre-denaturation at 94 °C for 2 sec, denaturation at 98 °C for 10 sec, annealing at 55 °C for 30 sec, extension at 68 °C for 30 sec, 35 cycles, and final extension at 72 °C for 5 min.

[0069] The nucleotide sequence of primer CRISPR-S-Aokap12-F is: GACTCCGTCGCGTATCGTCG (SEQ ID NO.8);

[0070] The nucleotide sequence of primer CRISPR-S-Aokap12-R is: AGCCACCGGGGATTCCAGTA (SEQ ID NO.9).

[0071] Directly sequence the original PCR product obtained by amplification (send it to General Biology (Anhui) Co., Ltd. for sequencing) to analyze the target sequence.

[0072] Through sequence alignment analysis, 2 Aokap12 gene-edited mutants, namely Aokap12 knockout strains (ΔAokap12-1 and ΔAokap12-2), were found. Among them, the Aokap12 knockout strain ΔAokap12-1 deleted 4 bases GAAG ( Figure 1 ) at the position 64 bp downstream of the translation start codon of the Aokap12 gene, and the Aokap12 knockout strain ΔAokap12-2 deleted 19 bases CACCGGAAGGTCCTGCGCC at the position 59 bp downstream of the translation start codon of the Aokap12 gene (see Figure 1),Both the Aokap12 gene knockout strains ΔAokap12-1 and ΔAokap12-2 resulted in frameshift mutations in the Aokap12 gene, causing premature termination of the transcript and forming polypeptides of 173 or 168 amino acids, while the full-length AoKap12 protein sequence contains 279 amino acids. Therefore, it is considered that the Aokap12 gene after gene editing may lose its function due to premature termination caused by frameshift mutations (see Figure 2 ).

[0073] Example 6: Determination of kojic acid production of Aokap12 gene-edited mutants

[0074] Inoculate the spore suspensions of Aspergillus oryzae 3.042 (WT), the Aokap12 gene knockout strain ΔAokap12-1, and the Aokap12 gene knockout strain ΔAokap12-2 on a solid kojic acid fermentation medium (100 g / L glucose, 1 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, 0.5 g / L potassium chloride, 1 g / L yeast extract) and culture for 3 days. Then add an appropriate amount of sterile water and collect the spore suspension. Count the number of spores in the spore suspension using a hemocytometer, and take 100 μL of the spore suspension (1×10 7 spores / mL) and inoculate it into 100 mL of liquid kojic acid fermentation medium. The fermentation conditions are 30 °C and 200 rpm. The kojic acid content is determined by the ferric sulfate colorimetric method, and the specific operation is as follows: After 7 days of fermentation, take 2 mL of the fermentation broth, centrifuge at 12000 rpm for 2 min, take 0.5 mL of the fermentation supernatant, add 0.5 mL of ddH2O and 1 mL of FeCl3-HCl colorimetric solution (0.06 M FeCl3 and 0.27 M HCl), and vortex to mix evenly. Use water to replace the fermentation supernatant as a control, measure the absorbance at a wavelength of 500 nm using a microplate reader, and finally calculate the kojic acid production of each strain through the prepared kojic acid standard curve.

[0075] Since kojic acid can form a red complex with Fe 3+ , kojic acid can be colored by FeCl3. The kojic acid color development results show that the fermentation broths of the two Aokap12 gene knockout strains showed a deeper red color than the fermentation broth of the wild-type Aspergillus oryzae 3.042 strain, indicating that knocking out Aokap12 can increase the kojic acid production of Aspergillus oryzae (see the kojic acid color development results in Figure 3as shown in A in). The kojic acid content determination shows that the kojic acid production of the Aoka12 knockout strain is 2.3 times that of the wild-type Aspergillus oryzae 3.042 strain, and the average content reaches 35.02 mg / ml·g. Among them, the kojic acid production of the wild-type Aspergillus oryzae 3.042 strain is 15.33 mg / ml·g, the kojic acid production of the Aokap12 knockout strain ΔAokap12-1 is 34.57 mg / ml·g, and the kojic acid production of the Aokap12 knockout strain ΔAokap12-2 is 35.46 mg / ml·g. The results of the kojic acid production determination are as Figure 3 shown in B in).

[0076] Obviously, the above-mentioned embodiments of the present invention are merely examples for more clearly explaining the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or variations can be made on the basis of the above description. It is impossible to enumerate all the implementation methods here. Any obvious changes or variations derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. A gene for increasing the kojic acid production of Aspergillus oryzae, characterized in that The gene is the Aspergillus oryzae Aokap12 gene; the Aspergillus oryzae Aokap12 gene has any one of the following nucleotide sequences: 1) the nucleotide sequence shown in SEQ ID NO.1; 2) A nucleotide sequence that has more than 90% homology with the nucleotide sequence shown in SEQ ID NO.1 and encodes the amino acid sequence shown in SEQ ID NO.

2.

2. Use of the Aspergillus oryzae Aokap12 gene as claimed in claim 1 in improving the kojic acid production of Aspergillus oryzae.

3. The use of the Aspergillus oryzae Aokap12 gene according to claim 2 in improving the kojic acid production of Aspergillus oryzae, characterized in that: The Aokap12 gene was knocked out in Aspergillus oryzae, and an Aokap12 gene-positive engineered bacterium was constructed, thereby increasing the kojic acid production of Aspergillus oryzae.

4. A recombinant vector for editing the gene of Aspergillus oryzae Aokap12, characterized in that: The Aspergillus oryzae Aokap12 gene editing recombinant vector is a recombinant vector constructed by using CRISPR / Cas9 technology to destroy the open reading frame of the Aokap12 gene; the Aspergillus oryzae Aokap12 gene editing recombinant vector contains the target sequence of the Aspergillus oryzae Aokap12 gene.

5. The Aspergillus oryzae Aokap12 gene editing recombinant vector according to claim 4, characterized in that The target sequence of the Aspergillus oryzae Aokap12 gene is shown in SEQ ID NO.

3.

6. A method for increasing the kojic acid production of Aspergillus oryzae, characterized in that: The following steps are involved: The Aspergillus oryzae Aokap12 gene editing recombinant vector as described in claim 4 is transferred into Aspergillus oryzae protoplasts to knock out the Aokap12 gene in Aspergillus oryzae.

7. The method for increasing kojic acid production of Aspergillus oryzae according to claim 6, characterized in that: The specific steps include: 1) Using the vector pPTRII-Cas9-kojA as a template, carrying the Aspergillus oryzae U6 promoter PU6, sgRNA and Aspergillus oryzae U6 terminator TU6 sequences, the Aspergillus oryzae U6 promoter PU6 was amplified with primers PU6-F and PU6-Aokap12-R to obtain the PU6-Aokap12 target fragment, and the sgRNA and Aspergillus oryzae U6 terminator TU6 were amplified with primers TU6-Aokap12-F and TU6-R to obtain the sgRNA-Aokap12 target fragment; 2) Using the PU6-Aokap12 target fragment and the sgRNA-Aokap12 target fragment as templates, overlapping PCR amplification was performed with primers PU6-F and TU6-R, and the sgRNA expression cassette containing the Aokap12 gene target sequence was obtained by purifying the PCR product; 3) digesting the pPTRII-Cas9 vector and purifying and recovering the linearized pPTRII-Cas9 vector, and recombining and connecting the linearized pPTRII-Cas9 vector with the PCR amplification product of the sgRNA expression cassette fragment purified and recovered in step 2) to obtain the Aspergillus oryzae Aokap12 gene editing recombinant vector; 4) The Aspergillus oryzae Aokap12 gene editing recombinant vector is transferred into Aspergillus oryzae protoplasts, the Aokap12 gene is knocked out in Aspergillus oryzae and identified to obtain Aokap12 gene knockout positive engineered bacteria.

8. The method for increasing kojic acid production of Aspergillus oryzae according to claim 7, characterized in that: The sequences of the primers used are as follows: Primer PU6-F: CGACTCTAGAGGATCCCCGGGTAATGCCGGCTCATTCAAA; Primer PU6-Aokap12-R: GAAGGTCCTGCGCCCACCGAACTTGTTCTTCTTTACAATGATTTATTTA; Primer TU6-Aokap12-F: TCGGTGGGCGCAGGACCTTCGTTTTAGAGCTAGAAATAGCAAGTTAAA; Primer TU6-R: AATTCGAGCTCGGTACCCGGGAGCAGCTCTATATCACGTGACG.

9. The method for increasing kojic acid production of Aspergillus oryzae according to claim 7, characterized in that: The sequences of the primers used to identify the gene editing status of the Aokap12 gene knockout positive engineered bacteria are as follows: Primer CRISPR-S-Aokap12-F: GACTCCGTCGCGTATCGTCG; Primer CRISPR-S-Aokap12-R: AGCCACCGGGGATTCCAGTA.

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