Aokap11 gene for improving production of kojic acid by aspergillus oryzae and application thereof

Abstract

This invention belongs to the field of microbial genetic engineering technology, specifically relating to a method for increasing the yield of kojic acid from Aspergillus oryzae. Aokap11 Genes and their applications. (The following is a separate section...) Aokap11 The gene has any of the following nucleotide sequences: 1) a nucleotide sequence as shown in SEQ ID NO.1; 2) a nucleotide sequence generated by adding, substituting, or deleting one or more bases from the nucleotide sequence shown in SEQ ID NO.1, and encoding a nucleotide sequence that affects the kojic acid synthesis function of the protein in *Aspergillus oryzae*. This invention utilizes gene editing technology to knock out [a specific gene] in the recipient bacterium *Aspergillus oryzae*. Aokap11 Genes, making Aokap11 Loss of gene function or weakened expression significantly increases kojic acid production in Aspergillus oryzae.

Description

Technical Field

[0001] This invention belongs to the field of microbial genetic engineering technology, specifically relating to the Aokap11 gene for increasing kojic acid production from Aspergillus oryzae and its application. Background Technology

[0002] Kojic acid is an organic acid mainly produced by the fermentation of Aspergillus oryzae. It possesses various biological activities, including antibacterial, antioxidant, and anti-tyrosinase properties, and has wide applications in food, pharmaceuticals, cosmetics, and agriculture. The kojic acid market size was US$35 million in 2020, and with the increasing maturity of the kojic acid and its derivatives market, the demand for kojic acid will continue to grow. Therefore, kojic acid is a product with great market potential, and achieving efficient synthesis of kojic acid is an important aspect of kojic acid research.

[0003] Currently, kojic acid fermentation primarily uses *Aspergillus oryzae* strains as industrial production strains. However, the kojic acid yield of *Aspergillus oryzae* strains isolated from nature is insufficient to meet the ever-increasing demand for kojic acid. The current solution involves modifying *Aspergillus oryzae* using mutagenesis and genetic engineering techniques to obtain high-yield kojic acid strains. However, genetic modification presupposes the cloning of genes involved in kojic acid synthesis regulation; therefore, identifying gene targets to increase kojic acid production in *Aspergillus oryzae* is crucial for achieving efficient kojic acid synthesis. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide an Aokap11 gene for increasing kojic acid production from Aspergillus oryzae and its application.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] This invention provides an Aokap11 gene for increasing kojic acid production from Aspergillus oryzae, wherein the Aokap11 gene has any one of the following nucleotide sequences:

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

[0008] 2) The nucleotide sequence shown in SEQ ID NO.1 is generated by adding, substituting or deleting one or more bases and encodes a nucleotide sequence that affects the functional protein that affects the synthesis of kojic acid from Aspergillus oryzae.

[0009] Preferably, the nucleotide sequence of the functional protein affecting kojic acid synthesis in Aspergillus oryzae is shown in SEQ ID NO.3. The nucleotide sequence shown in SEQ ID NO.3, a target sequence based on CRISPR / Cas9 technology, can inhibit the expression or activity of the Aokap11 gene, causing loss of Aokap11 gene function or reduced expression, thereby increasing the kojic acid yield of Aspergillus oryzae.

[0010] The present invention also provides the application of the aforementioned Aokap11 gene in increasing the yield of kojic acid from Aspergillus oryzae.

[0011] As a preferred embodiment of the application described in this invention, the application uses gene editing technology to knock out the Aokap11 gene in Aspergillus oryzae, thereby disrupting the open reading frame of the Aokap11 gene in Aspergillus oryzae and thus increasing the kojic acid production of Aspergillus oryzae.

[0012] As a preferred embodiment of the application described in this invention, the gene editing technology includes CRISPR / Cas9 technology.

[0013] The present invention also provides a method for constructing a high-kojic acid-producing Aspergillus oryzae, wherein the method involves using gene editing technology to knock out the Aokap11 gene in Aspergillus oryzae.

[0014] Preferably, the method involves knocking out the Aokap11 gene in Aspergillus oryzae using a CRISPR / Cas9 recombinant vector; the CRISPR / Cas9 recombinant vector is a pPTRII-Cas9-Aokap11 vector containing a sgRNA that specifically targets the Aokap11 gene; the nucleotide sequence of the sgRNA that specifically targets the Aokap11 gene is shown in SEQ ID NO.3.

[0015] Preferably, the specific steps for knocking out the Aokap11 gene in Aspergillus oryzae using gene editing technology are as follows:

[0016] 1) Select a target sequence from the Aokap11 gene to increase kojic acid production from Aspergillus oryzae; the nucleotide sequence of the target sequence of the Aokap11 gene is shown in SEQ ID NO.3;

[0017] 2) Synthesize primers PU6-Aokap11-R and TU6-Aokap11-F containing the target sequence of the Aokap11 gene;

[0018] 3) Using pPTRII-Cas9-kojA plasmid as a template, the Aspergillus oryzae U6 promoter PU6 was amplified using primers PU6-F and PU6-Aokap11-R to obtain the PU6-Aokap11 target sequence; and the sgRNA and Aspergillus oryzae U6 terminator TU6 were amplified using primers TU6-Aokap11-F and TU6-R to obtain the sgRNA-Aokap11 target sequence.

[0019] 4) Overlap PCR amplification of the PU6-Aokap11 target sequence and the sgRNA-Aokap11 target sequence was performed using primers PU6-F and TU6-R to obtain the PU6-Aokap11-sgRNA-TU6 expression cassette fragment containing the Aspergillus oryzae U6 promoter PU6, the Aokap11 gene target sequence, sgRNA, and the Aspergillus oryzae U6 terminator TU6;

[0020] 5) The linearized pPTRII-Cas9 vector was recombined with the PU6-Aokap11-sgRNA-TU6 expression cassette fragment to obtain a CRISPR / Cas9 recombinant vector for knocking out the Aokap11 gene.

[0021] 6) The CRISPR / Cas9 recombinant vector was introduced into the recipient Aspergillus oryzae to obtain an engineered strain of Aspergillus oryzae that produces high levels of kojic acid.

[0022] Preferably, the CRISPR / Cas9 recombinant vector is introduced into the recipient Aspergillus oryzae using conventional biotechnological methods such as Agrobacterium-mediated transformation, Ti plasmids, or direct DNA transformation.

[0023] More preferably, the receptor Aspergillus oryzae is Aspergillus oryzae 3.042.

[0024] More preferably, in step 2), the sequence of primer PU6-Aokap11-R is shown in SEQ ID NO.4, and the sequence of primer TU6-Aokap11-F is shown in SEQ ID NO.5; in step 3), the sequence of primer PU6-F is shown in SEQ ID NO.6, and the sequence of primer TU6-R is shown in SEQ ID NO.7.

[0025] More preferably, the knockout of the Aokap11 gene in Aspergillus oryzae using gene editing technology further includes amplifying the target region of the Aokap11 gene in the Aspergillus oryzae engineered strain obtained in step 6) using primers CRISPR-S-Aokap11-F and CRISPR-S-Aokap11-R, thereby obtaining an Aokap11 gene knockout engineered strain; wherein:

[0026] The sequence of primer CRISPR-S-Aokap11-F is shown in SEQ ID NO.10;

[0027] The sequence of primer CRISPR-S-Aokap11-R is shown in SEQ ID NO.11.

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

[0029] (1) This invention provides a novel gene capable of increasing kojic acid production from Aspergillus oryzae: the Aokap11 gene. By knocking out the Aokap11 gene in the recipient strain Aspergillus oryzae using gene editing technology, the gene's function is lost or its expression level is reduced, thereby significantly increasing kojic acid production from Aspergillus oryzae. This invention provides a new gene resource for increasing kojic acid production from Aspergillus oryzae.

[0030] (2) This invention provides a method for efficient site-directed knockout of the Aokap11 gene. This invention discovers that using the nucleotide sequence (SEQ ID NO.3) downstream of the translation codon start site of the Aspergillus oryzae Aokap11 gene as the target sequence can achieve efficient site-directed knockout of the Aokap11 gene, thereby improving the efficiency of constructing Aspergillus oryzae engineered bacteria with high kojic acid production using the Aokap11 gene mutation. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the mutation types of the Aokap11 gene nucleotide sequence in a homozygous mutant strain of the Aokap11 gene in the wild-type Aspergillus oryzae 3.042 background.

[0032] Figure 2 This is a schematic diagram of the mutation types of the Aokap11 gene amino acid sequence in a homozygous mutant strain of the Aokap11 gene in the wild-type Aspergillus oryzae 3.042 background.

[0033] Figure 3 This is a graph showing the kojic acid production analysis results of Aspergillus oryzae 3.042 and Aokap11 mutant strains. Detailed Implementation

[0034] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. The embodiments given are merely illustrative of the invention and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0036] The Aspergillus strain used in the following examples, Aspergillus oryzae 3.042 (CICC 40092), is available to the public from the China Industrial Microbial Culture Collection Center.

[0037] In this embodiment of the 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. This biomaterial is available to the public from the School of Life Sciences, Jiangxi University of Science and Technology. It is intended solely for repeating experiments related to this invention and should not be used for other purposes.

[0038] Example 1: Gene Alignment

[0039] The nucleotide sequence (CDS sequence) of the AoKap11 gene was obtained from NCBI, as shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0040] SEQ ID NO.1:

[0041]

[0042] SEQ ID NO.2:

[0043] MRILAINLILAASALALDQAPLQLQDSSSGNIPDIRNNDTHGRPNIVFILVDDQDLQMDSLSYTPHTNHYIRDQGVFYKNHFVTTALCCPSRVSLWTGKQAHNTNVTEIYPPYGGYPKFVSEGHNENWLPLWLQDAGYNTYYTGKLFN AHTVDNYNLPFAKGFNTSDFVLDPYTYQYLHPVYQRNHDPPISYSGQHTIDVLRKKALDLLDDAVAESHERPFFLTIAPIAPHSNFEMTNASDYTTFRFSAPIPLERHKDLFPEVKVPRTEHFNPDQPSGVNWISTLPQQNQSSIDSNDE FYRARLRALQGVDEIVEQIVQRLEDAGVLDNTYIFYTSDNGYHIGQHRLHPGKECGFEEDIRVPMFIRGPGIPSGEEVGFVTTHIDLAPTIFEIAGLDLKEEFDGTPVPLTARDVEEEKLSKGRHEHVNVEYWGKAGFEGEMSRAPDGG PIAFRNNTYKALRVLGEGYNLYYSVWCTNEHELYDLTTDPYELNNLHPSVSGNIEQKLLGYPIQKVISRLDALLLVLKSCKGLTCVKPWEVLHPAGDVQTLSDALDDVFDVFYDEQIKVEYGWCEEGYIVEAEGPQVPAIYQGTRWSDWW

[0044] Example 2: Construction of the Aokap11 gene knockout vector to increase kojic acid production from Aspergillus oryzae

[0045] 1) Design of Aokap11 gene target sequences

[0046] Using the CDS sequence of the Aokap11 gene from *Aspergillus oryzae* RIB40 as a reference, Cas9 target analysis was performed on the Aokap11 gene CDS sequence using the CRISPRdirect web server (https: / / crispr.dbcls.jp / ), and the Aokap11 gene target sequence was designed, the nucleotide sequence of which is shown in SEQ ID NO.3. Primers PU6-Aokap11-R and TU6-Aokap11-F, each containing the Aokap11 gene target sequence, were also designed. The 3' ends of primers PU6-Aokap11-R and TU6-Aokap11-F have 28-29 nt (nucleotides) that pair with the *Aspergillus oryzae* U6 promoter PU6 and sgRNA, respectively. The sequence of primer PU6-Aokap11-R is shown in SEQ ID NO.4, and the sequence of primer TU6-Aokap11-F is shown in SEQ ID NO.5.

[0047] SEQ ID NO.3: GGACATCATCCGCAACAATG

[0048] SEQ ID NO.4: GGACATCATCCGCAACAATGACTTGTTCTTCTTTACAATGATTTATTTA

[0049] SEQ ID NO.5: CATTGTTGCGGATGATGTCCGTTTTAGAGCTAGAAATAGCAAGTTAAA

[0050] 2) Constructing sgRNA expression cassettes via overlap PCR

[0051] Using pPTRII-Cas9-kojA plasmid as a template (pPTRII-Cas9-kojA plasmid has been described in Yuzhen Li's book) # Huanxin Zhang # Junxia Fan #Ziming Chen, Tianming Chen, Bin Zeng, Zhang He* 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 pPTRII-Cas9-kojA plasmid contains the Aspergillus oryzae U6 promoter PU6, sgRNA and Aspergillus oryzae U6 terminator TU6 sequence. The Aspergillus oryzae U6 promoter PU6 was amplified using primers PU6-F (SEQ ID NO. 6) and PU6-Aokap11-R (SEQ ID NO. 4) to obtain the PU6-Aokap11 target sequence containing the Aspergillus oryzae U6 promoter PU6 and the Aokap11 gene target sequence; the primers TU6-Aokap11-F (SEQ ID NO. 5) and TU6-R (SEQ ID NO. 4) were then used to amplify the target sequence. NO.7) sgRNA and the Aspergillus oryzae U6 terminator TU6 were amplified to obtain the sgRNA-Aokap11 target sequence containing the Aokap11 gene target sequence, sgRNA, and the Aspergillus oryzae U6 terminator TU6. The amplification reaction system is shown in Table 1. The PCR reaction procedure is shown in Table 2.

[0052] SEQ ID NO.6: CGACTCTAGAGGATCCCCGGGTAATGCCGGCTCATTCAAA

[0053] SEQ ID NO.7: AATTCGAGCTCGGTACCCGGGAGCAGCTCTATATCACGTGACG

[0054] Table 1. 50 μL amplification reaction system

[0055] Components volume Aspergillus oryzae RIB40 DNA 1μL Primers (10 μM) 4μL dNTPs (10mM each) 1μL 5×Buffer 10μL Enzyme 1μL <![CDATA[ddH2O]]> 33μL

[0056] Table 2 PCR reaction procedures

[0057] step temperature time Pre-variation 95℃ 3min transsexual 95℃ 15 seconds annealing 55℃ 15 seconds extend 72℃ 1kb / 1min Complete extension 72℃ 5min

[0058] The PCR reaction program shown in Table 2 is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 1 min, 35 cycles; 72℃ complete extension for 5 min.

[0059] 1 μL of each of the PCR products containing the PU6-Aokap11 target sequence and the sgRNA-Aokap11 target sequence were taken and subjected to overlap PCR amplification using primers PU6-F (SEQ ID NO. 6) and TU6-R (SEQ ID NO. 7). The overlap PCR amplification reaction system is shown in Table 3. The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 1 min, 35 cycles; 72℃ final extension for 5 min. The amplified PCR products were detected by electrophoresis. The PCR product recovery yielded the PU6-Aokap11-sgRNA-TU6 expression cassette fragment containing the Aspergillus oryzae U6 promoter PU6, the Aokap11 gene target sequence, sgRNA, and the Aspergillus oryzae U6 terminator TU6, thus obtaining the constructed sgRNA expression cassette.

[0060] Table 3. 50 μL amplification reaction system for overlap PCR

[0061] Components volume PCR product of PU6-Aokap11 target sequence 1μL PCR product of sgRNA-Aokap11 target sequence 1μL Primer PU6-F (10 μM) 2μL Primer TU6-R (10 μM) 2μL dNTPs (10mM each) 1μL 5×Buffer 10μL Enzyme 1μL <![CDATA[ddH2O]]> 32μL

[0062] 3) The constructed sgRNA expression cassette was recombinantly ligated into the Cas9 vector.

[0063] The pPTRII-Cas9 vector was digested with the restriction endonuclease Sma I (37℃, 1h). The digestion system was: 2 μg pPTRII-Cas9 vector, 5 μL 10×QuickCut Green Buffer, 2 μL Sma I, and ddH2O added to a final volume of 50 μL. After confirming the digestion by agarose gel electrophoresis, the linearized pPTRII-Cas9 vector was recovered using a PCR product recovery kit (Novizan). Then, the linearized pPTRII-Cas9 vector was recombinated and ligated with the PU6-Aokap11-sgRNA-TU6 expression cassette fragment using a seamless cloning kit (Novizan). The specific recombination and ligation procedure is shown in Table 4.

[0064] Table 4 Recombinant Linkage Reaction System

[0065]

[0066] The reaction was carried out at 37°C for 30 min to obtain the ligation product pPTRII-Cas9-Aokap11 vector, which is the CRISPR / Cas9 recombinant vector used to knock out the Aokap11 gene.

[0067] 4) Transformation and identification of Escherichia coli

[0068] 2.5 μL of the ligation product was added to freshly thawed *E. coli* DH5α strain (Full Gold), and transformation was performed using the heat shock method. Positive clones were screened on ampicillin-resistant LB agar plates. Single clones were selected and cultured at 37°C for 12 hours before PCR amplification. The primers used for PCR amplification were primer pPTRII-F (SEQ ID NO. 8) and primer pPTRII-R (SEQ ID NO. 9). The PCR amplification system and PCR procedure are shown in Tables 1 and 2, respectively. The target band was detected by electrophoresis at approximately 750 bp. Positive bacterial cultures were selected and cultured overnight at 37°C (12 hours). The pPTRII-Cas9-Aokap11 vector was extracted using a plasmid miniprep kit (Tiangen).

[0069] SEQ ID NO.8: TGCATGCCTGCAGGTCGACT

[0070] SEQ ID NO.9: AAGGGGGATGTGCTGCAAGG

[0071] Example 3: Construction of Aokap11 gene knockout engineered bacteria to increase kojic acid production from Aspergillus oryzae

[0072] 1) Protoplast transformation of Aspergillus oryzae

[0073] ① The PEG-CaCl2-mediated protoplast transformation method was used, with wild-type Aspergillus oryzae 3.042 (CICC 40092) strain as the genetic transformation recipient.

[0074] ② First, prepare Aspergillus oryzae protoplasts by lysing the Aspergillus oryzae mycelia cultured overnight (16-20 hours) with 1% Yatalase (TaKaRa) and 1.5% cell lysin (Sigma).

[0075] ③Then the pPTRII-Cas9-Aokap11 vector constructed above, Aspergillus oryzae protoplasts and PEG-CaCl2 solution were mixed and allowed to stand at room temperature in the dark for 20 min.

[0076] ④The reaction was then terminated with buffer (1.2M sorbitol, 50mM CaCl2·2H2O, 35mM NaCl, 10mM Tris-HCl, pH 7.5), and the protoplasts were collected by centrifugation.

[0077] ⑤ Finally, mix the protoplasts with M+Met medium containing 0.1 μg / ml pyridinethiamine (0.2% NH4Cl, 0.1% NH4SO4, 0.05% KCl, 0.05% NaCl, 0.1% KH2PO4, 0.05% MgSO4·7H2O, 0.002% FeSO4·7H2O, 2% glucose, 0.15% methionine, 1.2M sorbitol, 0.5% agar, pH 5.5), pour the mixture onto M+Met medium plates containing 0.1 μg / ml pyridinethiamine and 1.5% agar, and incubate at 30℃ for 3-5 days.

[0078] 2) Identification of Aokap11 gene knockout engineered bacteria

[0079] The mycelia obtained from the above transformation culture were transferred to CD medium containing 0.1 μg / ml pyridine thiamine (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). After incubation at 30℃ for 3-5 days, mycelia were picked and placed in centrifuge tubes, and 100 μL of 25 mM NaOH solution was added. The mixture was heated at 100℃ for 20 min. Using the supernatant as a template, the target region of the Aokap11 gene was amplified using primers CRISPR-S-Aokap11-F (SEQ ID NO. 10) and CRISPR-S-Aokap11-R (SEQ ID NO. 11). The enzyme used in the PCR amplification reaction was KOD DNA polymerase (TOYOBO). The amplification reaction system and PCR reaction procedure are shown in Tables 5 and 6.

[0080] SEQ ID NO.10: GGCTATTAACTTAATTCTGGCAGCGT

[0081] SEQ ID NO.11:ATCCACGGTGTGCGCGTTAA

[0082] Table 5 50μL amplification reaction system

[0083] Components volume template 2μL Primers (10 μM) 4μL dNTPs (2mM each) 4μL 2×Buffer 25μL DNA polymerase 1μL <![CDATA[ddH2O]]> 14μL

[0084] Table 6 PCR reaction procedure

[0085]

[0086] Sequencing analysis of the amplified PCR product stock solution revealed two Aokap11 mutant strains, namely Aokap11 gene knockout engineered bacteria (ΔAokap11-1 and ΔAokap11-2). The ΔAokap11-1 mutant exhibited an 11-base deletion 98 bp downstream of the Aokap11 gene start codon, with the deleted nucleotide being CGGATATCATT; the ΔAokap11-2 mutant exhibited a 1-base deletion 102 bp downstream of the Aokap11 gene start codon, with the deleted nucleotide being T (see [link to original text]). Figure 1 , Figure 1 The yellow arrow in the middle indicates the target sequence.

[0087] Example 4: Kojic acid production determination of the Aokap11 gene knockout engineered strain that increases kojic acid production from Aspergillus oryzae

[0088] Newly collected Aspergillus oryzae strain 3.042 (WT), Aokap11 gene knockout engineered strain ΔAokap11-1, and Aokap11 gene knockout engineered strain ΔAokap11-2 were inoculated into liquid 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). Fermentation was carried out at 30℃ and 200 rpm for 7 days. The kojic acid content was determined by the ferric sulfate colorimetric method. The specific procedure was as follows: 2 mL of fermentation broth was taken, centrifuged, and 500 μL of fermentation supernatant was aspirated. 500 μL of ddH2O and 1000 μL of FeCl3-HCl colorimetric solution (0.06 M FeCl3 and 0.27 M HCl) were added and mixed well. Water was used as a control instead of fermentation supernatant, and the absorbance was measured at a wavelength of 500 nm. The kojic acid concentration in the fermentation broth of each strain was calculated using the kojic acid standard curve. Simultaneously, collect the mycelia from the fermentation broth, dry them at 60℃ to constant weight, and determine the dry weight of the mycelia. The kojic acid yield of the strain is calculated by dividing the kojic acid concentration of the strain by the dry weight of the mycelia (mg / ml·g). Kojic acid and Fe 3+ Since kojic acid can form a red complex, its content can be qualitatively indicated by FeCl3 colorimetric assay. The kojic acid colorimetric results showed that the red color of the fermentation broth from the Aokap11 gene knockout engineered strain was significantly deeper than that of the wild-type Aspergillus oryzae strain 3.042, indicating that knocking out the Aokap11 gene can increase the kojic acid yield of Aspergillus oryzae (see kojic acid colorimetric results for details). Figure 3(As shown in A in the figure). The kojic acid yield determination results showed that the wild-type Aspergillus oryzae 3.042 strain in the control group had a kojic acid yield of 14.62 mg / ml·g. Compared with the wild-type Aspergillus oryzae 3.042 strain, the kojic acid yield of the Aokap11 gene knockout engineered strains ΔAokap11-1 and ΔAokap11-2 increased by more than 200%, reaching an average of 49.03 mg / ml·g. Specifically, the kojic acid yield of Aokap11 gene knockout engineered strain ΔAokap11-1 was 46.11 mg / ml·g, and the kojic acid yield of Aokap11 gene knockout engineered strain ΔAokap11-2 was 51.94 mg / ml·g (see [reference to kojic acid yield determination results]). Figure 3 (As shown in B in the figure).

[0089] Obviously, the above embodiments of the present invention are merely examples to illustrate the present invention more clearly, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all implementation methods here. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for increasing the yield of kojic acid from Aspergillus oryzae. Aokap11 The application of genes in increasing kojic acid production from Aspergillus oryzae is characterized by, Knockout in Aspergillus oryzae using CRISPR / Cas9 recombinant vector Aokap11 Genes, among which, specific targets Aokap11 The nucleotide sequence of the gene's sgRNA is shown in SEQ ID NO.3, thereby increasing the production of kojic acid from Aspergillus oryzae; Aokap11 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A method for constructing a high-kojic acid-producing Aspergillus oryzae strain, characterized in that, Knockout in Aspergillus oryzae using CRISPR / Cas9 recombinant vector Aokap11 Gene; the CRISPR / Cas9 recombinant vector contains specific targeted genes. Aokap11 The pPTRII-Cas9-Aokap11 vector for the sgRNA of the gene; the specific target Aokap11 The nucleotide sequence of the gene's sgRNA is shown in SEQ ID NO.3; Aokap11 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. The method for constructing a high-kojic acid-producing Aspergillus oryzae according to claim 2, characterized in that, Knockout in Aspergillus oryzae Aokap11 The specific steps of gene generation are as follows: 1) In increasing the yield of kojic acid from Aspergillus oryzae Aokap11 Target sequences were selected from the gene; among them, Aokap11 The nucleotide sequence of the gene target sequence is shown in SEQ ID NO.3; 2) Synthesis with Aokap11 Primers PU6-Aokap11-R and TU6-Aokap11-F for the gene target sequence; wherein, the sequence of primer PU6-Aokap11-R is shown in SEQ ID NO.4, and the sequence of primer TU6-Aokap11-F is shown in SEQ ID NO.5; 3) The Aspergillus oryzae U6 promoter PU6 was amplified using primers PU6-F and PU6-Aokap11-R to obtain PU6- Aokap11 Target sequence; and amplification of sgRNA and Aspergillus oryzae U6 terminator TU6 using primers TU6-Aokap11-F and TU6-R to obtain sgRNA- Aokap11 Target sequences; wherein the sequence of primer PU6-F is shown in SEQ ID NO.6, and the sequence of primer TU6-R is shown in SEQ ID NO.7; 4) Using primers PU6-F and TU6-R to target PU6- Aokap11 Target sequence and sgRNA- Aokap11 The target sequence was amplified by overlap PCR to obtain the PU6 sequence containing the Aspergillus oryzae U6 promoter. Aokap11 Gene target sequence, sgRNA, and the PU6-Aokap11-sgRNA-TU6 expression cassette fragment of Aspergillus oryzae U6 terminator TU6; 5) The linearized pPTRII-Cas9 vector was recombinated and ligated with the PU6-Aokap11-sgRNA-TU6 expression cassette fragment to obtain a knockout vector. Aokap11 CRISPR / Cas9 recombinant vectors for genes; 6) The CRISPR / Cas9 recombinant vector was introduced into the recipient Aspergillus oryzae to obtain an engineered strain of Aspergillus oryzae that produces high levels of kojic acid.

4. The method for constructing a high-kojic acid-producing Aspergillus oryzae according to claim 3, characterized in that, The recipient Aspergillus oryzae is Aspergillus oryzae 3.

042.

5. The method for constructing a high-kojic acid-producing Aspergillus oryzae according to claim 3, characterized in that, This also includes the Aspergillus oryzae engineered strains obtained in step 6) amplified using primers CRISPR-S-Aokap11-F and CRISPR-S-Aokap11-R. Aokap11 The target region of the gene, thereby obtaining Aokap11 Gene knockout engineered bacteria; among which: The sequence of primer CRISPR-S-Aokap11-F is shown in SEQ ID NO.10; The sequence of primer CRISPR-S-Aokap11-R is shown in SEQ ID NO.11.

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