Application of Aspergillus oryzae Aokap9 gene in increasing kojic acid yield of Aspergillus oryzae

Abstract

The invention relates to an application of an Aspergillus oryzae Aokap9 gene in improving the kojic acid yield of Aspergillus oryzae, which is characterized in that an expression cassette containing an Aspergillus oryzae U6 promoter PU6, an Aokap9 gene target sequence, sgRNA and an Aspergillus oryzae U6 terminator TU6 is constructed by inhibiting the expression or activity of the Aokap9 gene in the Aspergillus oryzae and taking the 278th site to the 297th site of the Aokap9 gene as target regions, and is inserted into a gene editing vector pPRTII-Cas9, so that the Aspergillus oryzae Aokap9 gene is obtained. The method comprises the following steps: generating a gene editing vector pPRTII-Cas9-Aokap9 by using aspergillus oryzae as a raw material, then carrying out protoplast genetic transformation on the aspergillus oryzae, screening strains with high yield of kojic acid, and improving the kojic acid yield of the aspergillus oryzae. According to the invention, the CRISPR / Cas9 technology is utilized to excavate and identify the gene for improving the kojic acid yield of aspergillus oryzae, and the aspergillus oryzae strain with high kojic acid yield is constructed, so that the efficient biosynthesis of the kojic acid of aspergillus oryzae is effectively promoted.

Description

Technical Field

[0001] This application relates to Aspergillus oryzae Aokap9 The field of gene application technology, specifically involving Aspergillus oryzae Aokap9 Application of genes in improving kojic acid production in Aspergillus oryzae. Background Art

[0002] Kojic acid, whose chemical name is 2-hydroxymethyl-5-hydroxy-4-pyrone, is an organic acid with multiple uses that is mainly produced by Aspergillus microorganisms using sugar fermentation. Kojic acid has the effects of inhibiting tyrosinase, preserving, preserving, and anti-oxidation, and is therefore widely used in industries such as cosmetics, food, and medicine. At present, the production of kojic acid is mainly produced by fermentation of Aspergillus oryzae with sugars such as glucose as raw materials. Therefore, obtaining high-yielding kojic acid strains has become the key to increasing kojic acid production. However, the kojic acid production of Aspergillus oryzae isolated from nature is generally not high, and it is usually necessary to obtain high-yielding kojic acid strains through mutagenesis screening. However, current studies have shown that the effect of mutagenesis in increasing kojic acid production is no longer obvious. Summary of the invention

[0003] The object of the present invention is to provide a kind of Aspergillus oryzae Aokap9 The application of genes in improving the kojic acid production of Aspergillus oryzae, using CRISPR / Cas9 technology to explore and identify genes that improve the kojic acid production of Aspergillus oryzae, construct a high-yielding kojic acid Aspergillus oryzae strain, and effectively promote the efficient biosynthesis of kojic acid in Aspergillus oryzae.

[0004] The technical solution adopted by the present invention is: an Aspergillus oryzae Aokap9 The application of the gene is specifically to introduce the Aspergillus oryzae Aokap9 Genes used to improve kojic acid production in Aspergillus oryzae.

[0005] Further, by inhibiting the Aokap9 Gene expression or activity, Aokap9 The 278th to 297th positions of the gene were selected as the target region to construct a gene containing Aspergillus oryzae U6 Promoter PU6, Aokap9 Gene target sequence, sgRNA and Aspergillus oryzae U6 The expression cassette of terminator TU6 was inserted into the gene editing vector pPRTII-Cas9 to generate the gene editing vector pPRTII-Cas9-Aokap9, and protoplast genetic transformation of Aspergillus oryzae was performed to screen strains with high kojic acid production, thereby increasing the kojic acid production of Aspergillus oryzae.

[0006] Furthermore, the Aspergillus oryzae Aokap9 Aspergillus oryzae Aokap9 The amino acid sequence of the gene is SEQ ID No.2:MAPSHLLCTFSPSRLTLFLSPPIFLLSSYLVICLSFAFRVYTFNSNTLVQIKTHLNNNTSTFIMSTTTTSVAASATASCNSVTYWQLPVDDAACALPKTGNYSDVMDK CCSPAKVTDFDNGCGLYCLAQGQSVGDLKDCLRKNGAQDGREFYCKGNDTATATASAPSSTKTGDKTGTATGSGASSTSSDSAAYAIQPAVSKGGLGLLAMVFCSALMGVVA.

[0007] Further, inhibiting the Aokap9 The method for screening high-producing kojic acid strains is to obtain the expression or activity of the gene by overlapping PCR technology. Aokap9 The target gRNA expression cassette PU6-Aokap9-sgRNA-TU6 is then placed Aokap9 The target gRNA expression box PU6-Aokap9-sgRNA-TU6 is connected to the gene editing vector pPRTII-Cas9 through recombination to obtain the gene editing vector pPTRII-Cas9-Aokap9, and the gene editing vector pPTRII-Cas9-Aokap9 is used to perform protoplast genetic transformation on Aspergillus oryzae to increase the kojic acid production of Aspergillus oryzae.

[0008] Furthermore, in the overlapping PCR technique, the gene editing vector pPRTII-Cas9-kojA is used as a template, and primers PU6-F and PU6-Aokap9-R are used for amplification to obtain a sequence containing Aokap9 The target PU6 fragment PU6-Aokap9; in addition, the gene editing vector pPRTII-Cas9-kojA was used as a template, and primers TU6-Aokap9-F and TU6-R were used for amplification to obtain a fragment containing Aokap9 The target gRNA and U6 terminator fragment Aokap9-sgRNA-TU6; Aokap9 The target fragment of PU6 is PU6-Aokap9 and contains Aokap9 The target gRNA and U6 terminator fragment Aokap9-sgRNA-TU6 were used as templates, and primers PU6-F and TU6-R were used for amplification to obtain a fragment containing Aokap9 sgRNA expression cassette for the target PU6-Aokap9-sgRNA-TU6.

[0009] Furthermore, the primers PU6-F and PU6- Aokap9 -R, Primer TU6- Aokap9 The sequences of primers TU6-F and TU6-R are as follows: PU6-F:CGACTCTAGAGGATCCCCGGGTAATGCCGGCTCATTCAAA; PU6-Aokap9-R: ATCTTTCTCCTGTCAAGCTAACTTGTTCTTCTTTACAATGATTTATTTA; TU6-Aokap9-F: TAGCTTGACAGGAGAAAGATGTTTTAGAGCTAGAAATAGCAAGTTAAA; TU6-R: AATTCGAGCTCGGTACCCGGGAGCAGCTCTATATCACGTGACG.

[0010] The beneficial effects of the present invention are: (1) The present invention is based on Aokap9 Gene as a new idea to increase the kojic acid content of Aspergillus oryzae through gene editing Aokap9 Gene can significantly increase the production of kojic acid in Aspergillus oryzae; experiments have shown Aokap9 The gene has the function of increasing the production of kojic acid in Aspergillus oryzae. Aokap9 The biological function of the gene-encoded protein can significantly increase the kojic acid yield of Aspergillus oryzae. The invention provides an important functional gene for genetic improvement of increasing the kojic acid yield of Aspergillus oryzae.

[0011] (2) The present invention uses gene editing technology to Aokap9 Aspergillus oryzae Aokap9 The nucleotide sequence from position 278 to 297 in the gene is used as the target sequence, which can effectively achieve Aokap9 Site-directed knockout of the gene, and insertion or deletion at positions 278 to 297 would result in Aokap9 The protein encoded by the gene is destroyed, which increases the production of kojic acid from Aspergillus oryzae and effectively improves the Aokap9 Efficiency of constructing engineered strains with gene mutations that produce high kojic acid production. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0013] Figure 1 In the wild-type Aspergillus oryzae 3.042 background Aokap9 Homozygous mutant strains Aokap9 Schematic diagram of mutation types in gene nucleotide sequences.

[0014] Figure 2 In the wild-type Aspergillus oryzae 3.042 background Aokap9 Homozygous mutant strains Aokap9 Schematic diagram of mutation types in gene amino acid sequences.

[0015] Figure 3 Aspergillus oryzae kojR Overexpression bacteria Aokap9 Homozygous mutant strains Aokap9 Schematic diagram of mutation types in gene nucleotide sequences.

[0016] Figure 4 Aspergillus oryzae kojR Overexpression bacteria Aokap9 Homozygous mutant strains Aokap9 Schematic diagram of mutation types in gene amino acid sequences.

[0017] Figure 5 Aspergillus oryzae 3.042, Aokap9 Mutant bacteria, Aspergillus oryzae kojR Aspergillus oryzae kojR Overexpression bacteria Aokap9 Kojic acid chromogenic diagram in the fermentation broth of the homozygous mutant strain.

[0018] Figure 6 Aspergillus oryzae 3.042, Aokap9 Mutant bacteria, Aspergillus oryzae kojR Aspergillus oryzae kojR Overexpression bacteria Aokap9 Determination of kojic acid content in homozygous mutant strains. DETAILED DESCRIPTION

[0019] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and therefore, the present invention is not limited to the limitations of the specific embodiments disclosed below.

[0020] The embodiment of the present invention provides a kind of Aspergillus oryzae Aokap9 The application of genes in improving the production of kojic acid in Aspergillus oryzae Aokap9 Gene expression or activity, Aokap9The target region is from position 278 to position 297 of the gene. Construct a gene containing Aspergillus oryzae U6 promoter PU6, Aokap9 The expression cassette of the gene target sequence, sgRNA and Aspergillus oryzae U6 terminator TU6 was inserted into the gene editing vector pPRTII-Cas9 to generate the gene editing vector pPRTII-Cas9-Aokap9, and the Aspergillus oryzae protoplasts were genetically transformed to screen strains with high kojic acid production to increase the kojic acid production of Aspergillus oryzae. Aokap9 The target gRNA expression cassette PU6-Aokap9-sgRNA-TU6 is then placed Aokap9 The target gRNA expression box PU6-Aokap9-sgRNA-TU6 is connected to the gene editing vector pPRTII-Cas9 through recombination to obtain the gene editing vector pPTRII-Cas9-Aokap9, and the gene editing vector pPTRII-Cas9-Aokap9 is used to perform protoplast genetic transformation on Aspergillus oryzae to increase the kojic acid production of Aspergillus oryzae.

[0021] In the overlapping PCR technique, the gene editing vector pPRTII-Cas9-kojA is used as a template, and primers PU6-F and PU6-Aokap9-R are used for amplification to obtain a Aokap9 The target PU6 fragment PU6-Aokap9; in addition, the gene editing vector pPRTII-Cas9-kojA was used as a template, and primers TU6-Aokap9-F and TU6-R were used for amplification to obtain a fragment containing Aokap9 The target gRNA and U6 terminator fragment Aokap9-sgRNA-TU6; Aokap9 The target fragment of PU6 is PU6-Aokap9 and contains Aokap9 The target gRNA and U6 terminator fragment Aokap9-sgRNA-TU6 were used as templates, and primers PU6-F and TU6-R were used for amplification to obtain a fragment containing Aokap9 sgRNA expression cassette for the target PU6-Aokap9-sgRNA-TU6.

[0022] The experimental methods in the examples of the present invention are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the examples of the present invention are commercially available unless otherwise specified.

[0023] The Aspergillus oryzae strain in the embodiment of the present invention is Aspergillus oryzae 3.042 (CICC 40092), which is available to the public from the China Industrial Microbiological Culture Collection Center. The CRISPR-Cas9 vector in the embodiment of the present invention is pPRTII-Cas9, which has been recorded in the document "A highly efficient identification of mutants generated by CRISPR / Cas9 using the non-functional DsRed assisted selection in vitro" published by Yuzhen Li, Huanxin Zhang, Junxia Fan, Ziming Chen, Tianming Chen, Bin Zeng and Zhe Zhang in 2021. Aspergillus oryzae , World Journal of Microbiology and Biotechnology 》, the public can obtain it from the School of Life Sciences of Jiangxi Science & Technology Normal University. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0024] Example 1 Aokap9 Construction of gene editing vector The gene editing vector used in the embodiment of the present invention is pPRTII-Cas9. The embodiment of the present invention designs the target site on the primer and then obtains the gene editing vector containing pPRTII-Cas9 by overlapping PCR. Aokap9 The target gRNA expression cassette PU6-Aokap9-sgRNA-TU6 is then connected to the pPRTII-Cas9 vector through homologous recombination. The specific construction process is as follows: (1) Design of target gRNA Will Aokap9 The gene sequence of gene (AO090011000266) was input into https: / / crispr.dbcls.jp for target design. Aokap9The nucleotide sequence of the gene is SEQ ID No.1: ATGGCACCAAGTCACCTGCTGTGTACTTTTAGTCCTTCGTATGTAACCGGGATCAATATAAATAAAACCCAATCACAATGGAACAAGCGAGACAGGGATTGCCGATCACCAATCGGGCTCCCGACGCCAAGCCAGAACGAGCGAGTGGAGCAAGGCGCGAGAACCAATTAGCGTTGAGCCGGTCAATCCTGGTTGTCAGCCCACAGCGTTCTTTGTATTGTCATATAAAGCTCTTTTTGACTTTCCCAGCCGTCTCACCTTGTTTCTTTCACCTCCTATCTTTCTCCTGTCAAGCTATCTTGTCATCTGTCTTTCGTTTGCATTTCGAGTCTACACTTTCAACTCCAATACACTCGTTCAAATCAAGACTCACTTGAACAACAACACAAGTACATTCATAATGTCTACTACCACCACCTCCGTCGCCGCCAGCGCCACTGCCTCCTGCAACAGCGTGACCTACTGGCAGCTCCCCGTCGACGATGCCGCCTGCGCCCTCCCCAAGACCGGCAACTACTCTGATGTCATGGACAAATGCTGCTCCCCGGCCAAGGTGACCGATTTCGACAACGGCTGCGGCCTCTACTGTCTCGCCCAGGGCCAGAGCGTCGGCGACCTCAAGGACTGCCTGCGCAAGAACGGCGCCCAGGACGGTCGCGAGTTCTACTGCAAGGGCAATGATACCGCCACCGCCACGGCCTCCGCTCCGTCTTCCACCAAGACCGGCGACAAGACCGGTACTGCCACTGGCAGCGGTGCCTCCTCCACCTCCTCCGACAGCGCCGCCTATGCTATCCAGCCTGCTGTTTCCAAGGGCGGACTCGGTCTCTTGGCCATGGTCTTCTGCTCCGCTTTGATGGGGGTTGTTGCCTAGATACTTTTCACTTGGGGTTGGCTACCTGGGAAGAATGGGTGGATCTGTCTATATGCTTTGTTTTCTTTGGTGATCCGTCTTGCGATGCATCCGGCAGGACATATTATTTAATACCCTTACGATATGAGCCTGCTGTATTATACTTTTTCTCGTGGTTTGGACGTCGGTCGGTATTGAGCTTGGTTATTCTAATTTTAATGACTGAAAAGTTCGATCTTTTATCTGA AACGTTCTGAAACTTGTTGCATTCCGTAGATTTGGAAACTTGAACCCGAGGAACCGTTATATTTTCCAGATAAG, the amino acid sequence is SEQ ID No.2: MAPSHLLCTFSPSRLTLFLS PPIFLLSSYLVICLSFAFRVYTFNSNTLVQIKTHLNNNTSTFIMSTTTTSVAASATASCNSVTYWQLPVDDAACALPKTGNYSDVMDKCCSPAKVTDFDNGCGLYCLAQGQSVGDLKDCLRKNGAQDGREFYCKGNDTATATASAPSSTKTGDKTGTATGSGASSTSSDSAAYAIQPAVSKGGLGLLAMVFCSALMGVVA. The DNA sequence of the target region selected in the embodiment of the present invention is Aspergillus oryzae Aokap9 The DNA sequence of the target region is Aspergillus oryzae Aokap9The nucleotide sequence from position 278 to position 297 in the gene, the Aspergillus oryzae U6 promoter PU6, sgRNA, and the Aspergillus oryzae U6 terminator TU6 sequences can be obtained from the paper "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 》 was obtained by direct amplification of the gene editing vector pPTRII-Cas9-kojA disclosed in

[0025] (2) Obtaining Aokap9 gRNA expression cassette for target PU6-Aokap9-sgRNA-TU6 ① Get included Aokap9 PU6 fragment of the target The gene editing vector pPTRII-Cas9-kojA was used as a template and primers PU6-F and PU6-Aokap9-R were used for amplification to obtain Aokap9 The target PU6 fragment PU6-Aokap9, the product length is 891 bp. The PCR system is as follows: template DNA (gene editing vector pPTRII-Cas9-kojA ≥ 30 ng / μl) 1.0 μl; primer PU6-F and primer PU6-Aokap9-R: 2 μl each; deoxyribonucleoside triphosphate dNTPs: 1.0 μl; 5× enzyme buffer buffer: 10 μl; double distilled water ddH2O: 33 μl; DNA polymerase (Vazyme product number: P501-d1): 1 μl. The PCR amplification program is as follows: 95℃ pre-denaturation for 30 seconds, 95℃ denaturation for 10 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 5 minutes, 35 cycles, 72℃ thorough extension for 5 minutes. The sequence of primer PU6-F is SEQ ID NO.3: CGACTCTAGAGGATCCCCGGGTAATGCCGGCTCATTCAAA, and the sequence of primer PU6-Aokap9-R is SEQ ID NO.4: ATCTTTCTCCTGTCAAGCTAACTTGTTCTTCTTTACAATGATTTATTTA.

[0026] ② Get included Aokap9Target gRNA and U6 terminator fragment The gene editing vector pPTRII-Cas9-kojA was used as a template and PCR amplification was performed using primers TU6-Aokap9-F and TU6-R to obtain a Aokap9 The target gRNA and U6 terminator fragment Aokap9-sgRNA-TU6. PCR system and amplification include Aokap9 The PCR system of the target PU6 fragment PU6-Aokap9 is the same. The PCR amplification program is as follows: 95°C pre-denaturation for 30 seconds, 95°C denaturation for 10 seconds, 55°C annealing for 30 seconds, 72°C extension for 5 minutes, 35 cycles, 72°C full extension for 5 minutes, the sequence of primer TU6-Aokap9-F is SEQ ID NO.5: TAGCTTGACAGGAGAAAGATGTTTTAGAGCTAGAAATAGCAAGTTAAA, and the sequence of primer TU6-R is SEQ ID NO.6: AATTCGAGCTCGGTACCCGGGAGCAGCTCTATATCACGTGACG.

[0027] ③ Obtain Aokap9 gRNA expression cassette for target To include Aokap9 The target fragment of PU6 is PU6-Aokap9 and contains Aokap9 The target gRNA and U6 terminator fragment Aokap9-sgRNA-TU6 were used as templates, and primers PU6-F and TU6-R were used for amplification to obtain a fragment containing Aokap9 The target sgRNA expression cassette PU6-Aokap9-sgRNA-TU6. The overlapping PCR amplification system is as follows: Aokap9 PU6-Aokap9 1.0 μl; contains Aokap9 Target gRNA and U6 terminator fragment Aokap9-sgRNA-TU6 1.0 μl; deoxyribonucleoside triphosphate dNTPs: 1.0 μl; 5× enzyme buffer: 10 μl; double distilled water ddH2O: 33 μl; DNA polymerase (Vazyme product number: P501-d1): 1 μl. The PCR amplification program is as follows: 95℃ pre-denaturation for 30 seconds, 95℃ denaturation for 10 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 5 minutes, after 5 cycles, add 4 μl of primer PU6-F and primer TU6-R mixture, the concentration is 10 μM. Continue amplification for 30 cycles to obtain a fragment containing Aokap9 The target sgRNA expression cassette PU6-Aokap9-sgRNA-TU6, and finally the PCR product is recovered.

[0028] (3) PU6-Aokap9-sgRNA-TU6 was connected to the pPRTII-Cas9 vector by recombination The pPRTII-Cas9 vector was digested with restriction endonuclease Sma I to recover the linear pPTRII-Cas9 fragment. The enzyme digestion system was as follows: pPRTII-Cas9 vector (250 ng / μl): 20 μl; 10× enzyme digestion buffer: 5 μl; restriction endonuclease Sma I (produced by Takara Bio): 2 μl; double distilled water ddH2O: 23 μL. Incubate at 37°C for 1 hour.

[0029] The linearized pPTRII-Cas9 fragment was ligated with the Aokap9 The target sgRNA expression cassette PU6-Aokap9-sgRNA-TU6 was recombined and connected to the pPRTII-Cas9 vector to obtain the gene editing vector pPTRII-Cas9-Aokap9. The recombination and connection reaction system is as follows: linearized pPRTII-Cas9 vector: 200 ng; PU6-Aokap9-sgRNA -TU6 fragment: 80 ng; 5× enzyme digestion buffer: 2 μl; recombinase Exnase II (Vazyme product number: C112-02): 2 μl; double distilled water ddH2O is added to the total volume of 10 μl. Incubate at 37°C for 30 minutes, and after the reaction, cool to 4°C or immediately place on ice to cool. Then obtain it through heat shock transformation of Escherichia coli and identification of transformants. Aokap9 Gene editing vector pPTRII-Cas9-Aokap9.

[0030] Example 2 Genetic transformation of Aspergillus oryzae protoplasts First, the newly harvested Aspergillus oryzae 3.042 and Aspergillus oryzae 3.042 were cultured in DPY liquid medium containing 2% glucose, 1% peptone, 0.5% yeast extract, 0.5% KH2PO4 and 0.05% MgSO4⋅7H2O at a pH of 5.5. kojRSpores of overexpressing bacteria were collected after 16-20 hours. Mycelia were then lysed with 1% filamentous fungal protoplast preparation enzyme Yatalase (produced by Bao Bio) and 1.5% cell lysis enzyme (produced by Sigma) to prepare protoplasts. 10 μg of gene editing vector pPTRII-Cas9-Aokap9 was mixed with 200 μl of protoplasts and incubated on ice for 30 minutes. Then, PEG buffer containing 60% polyethylene glycol PEG 4000, 50mM CaCl2⋅2H2O and 10 mM Tris-HCl, pH 7.5 was added and gently mixed. After standing at room temperature for 10-20 minutes, the PEG-treated protoplasts were diluted with a washing buffer containing 1.2 M sorbitol, 50 mM CaCl2⋅2H2O, 35 mM NaCl and 10 mM Tris-HCl, pH 7.5, and the protoplasts were collected by centrifugation at 4°C. Then, add M+Met medium containing 1.2 M sorbitol and 0.5% agar, mix well, spread on M+Met medium plate containing 1.2 M sorbitol and 1.5% agar, and culture at 30°C for 3-5 days. The M+Met medium includes 0.2% NH4Cl, 0.1% (NH4)2SO4, 0.05% KCl, 0.05% NaCl, 0.1% KH2PO4, 0.05% MgSO4⋅7H2O, 0.002% FeSO4⋅7H2O, 2% glucose, 0.15% methionine and 1.2M sorbitol, and the pH value is 5.5. After mycelium grows, transfer the mycelium to CD medium containing 0.1 μg / ml pyrithiamine, culture at 30°C for 3-5 days, and screen positive strains. The CD medium includes 2% glucose, 0.2% NaNO3, 0.1% KH2PO4, 0.05% MgSO4, 0.05% KCl, 0.05% NaCl and 0.002% FeSO4, with a pH of 5.5.

[0031] Example 3 Detection of Aspergillus oryzae CRISPR / Cas9 mutation results To determine the results of CRISPR / Cas9 mutations, PCR amplification and sequencing were used for target targeting. First, the mycelium was picked and placed in a centrifuge tube, 100 μl of 25 mM NaOH solution was added, and heated at 100°C for 20 minutes. The supernatant was used as a PCR template.

[0032] according to Aokap9Primers CRISPR-S-Aokap9-F and CRISPR-S-Aokap9-R were designed based on the gene sequence (AO090011000266). The product size was 542 bp. Primers CRISPR-S-Aokap9-F and CRISPR-S-Aokap9-R were used to amplify the gene using KOD DNA polymerase (manufacturer: Toyobo). Aokap9 Target region sequence, sequencing analysis Aokap9 Targeting sequence: The sequence of primer CRISPR-S-Aokap9-F is SEQ ID NO.7: AGACAGGGATTGCCGATCAC, and the sequence of primer CRISPR-S-Aokap9-R is SEQ ID NO.8: CAGGCAGTCC TTGAGGTCG.

[0033] Reaction system: supernatant DNA template: 2 μl; primer CRISPR-S-Aokap9-F and primer CRISPR-S-Aokap9-R mixture: 4 μl; deoxyribonucleoside triphosphate dNTPs: 4 μl; 2× enzyme buffer: 25 μl; KOD DNA polymerase: 1 μl; double distilled water ddH2O: 14 μl.

[0034] The PCR amplification program was as follows: pre-denaturation at 94°C for 2 seconds, denaturation at 98°C for 10 seconds, annealing at 55°C for 30 seconds, extension at 68°C for 30 seconds, 35 cycles, and complete extension at 72°C for 5 minutes.

[0035] The amplified PCR product stock solution was directly sent for testing. By sequencing the DNA sequences of the target regions of multiple positive transformation events, it was determined whether gene editing occurred in the target region. Finally, it was found that the target region sequences of the positive strains had changed. The sequences before and after editing were as follows: Figure 1 and Figure 3 shown.

[0036] right Aokap9 The targeted nucleotide sequence of the gene was sequenced and compared with the unedited wild-type WT. Aokap9 In mutant Aokap9 Genes in Figure 1 A 1-base insertion occurred at the target indicated, Aspergillus oryzae kojR Overexpression bacteria Aokap9 Gene homozygous mutant strain Δ Aokap9 -OE- kojR exist Aokap9 Genes in Figure 3 11 base deletion mutations occurred at the target site shown. These mutations caused the amino acids to change as follows Figure 2 or Figure 4, so Δ Aokap9 and Δ Aokap9 -OE- kojR Engineering bacteria Aokap9 The protein functions of the genes were all missing.

[0037] Example 4 Aspergillus oryzae Aokap9 Determination of kojic acid production in knockout engineered bacteria Aspergillus oryzae constructed by the present invention Aokap9 The method for producing kojic acid by fermentation using engineered bacteria comprises the following specific steps: Fermentation medium: 100 g / L glucose, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 0.5 g / L potassium chloride, 1 g / L yeast extract.

[0038] Fermentation method: Aspergillus oryzae 3.042, Aokap9 Knockout bacteria Δ Aokap9 , kojR Overexpression of OE- kojR and Δ Aokap9 -OE- kojR The bacteria were inoculated in the solid fermentation medium and cultured for 3 days. Then, an appropriate amount of sterile water was added to wash off the spores. The number of spores in the spore solution was counted using a hemocytometer, and 1×10 7 spores / mL were inoculated into the fermentation medium, the culture conditions were 28°C, 200 rpm, and the fermentation time was 7 days.

[0039] Determination method of kojic acid: The ferrous sulfate colorimetric method is used. Remove the mycelium from the fermentation broth by centrifugation, take 0.5 mL of the fermentation supernatant, add 0.5 mL of ddH2O and 1 mL of FeCl3-HCl colorimetric solution, and vortex to mix. The FeCl3-HCl colorimetric solution includes 0.06 M FeCl3 and 0.27 M HCl. Replace the fermentation supernatant with water as a control, and measure its absorbance at a wavelength of 500 nm. Calculate the kojic acid content of the sample using the kojic acid standard curve.

[0040] Kojic acid color development results are as follows Figure 5 As shown, the fermentation broth of the wild-type 3.042 strain is red. Aokap9 The knockout bacterial fermentation broth showed a darker red color, indicating that the knockout Aokap9 It can promote the synthesis of kojic acid in Aspergillus oryzae. Figure 6 As shown, the kojic acid content determination showed that Aokap9 The kojic acid production of the knockout strain was 1.6 times that of the wild-type 3.042 strain, with an average content of 23.67 mg / mlg. kojR Knockout in overexpressing bacteria Aokap9 Can significantly increase the amount of kojic acid synthesis in Aspergillus oryzae, Δ Aokap9 -OE-kojR The kojic acid production of the engineered bacteria increased by 131% compared with the wild type. kojR The overexpression bacteria increased by 25% to 33.16 mg / mlg.

[0041] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A type of Aspergillus oryzae Aokap9 The application of genes is characterized in that Aspergillus oryzae Aokap9 Genes used to improve kojic acid production in Aspergillus oryzae.

2. A kind of Aspergillus oryzae according to claim 1 Aokap9 The application of genes is characterized in that By inhibiting Aspergillus oryzae Aokap9 Gene expression or activity, Aokap9 The 278th to 297th position of the gene was taken as the target region, and a plasmid containing Aspergillus oryzae U6 promoter PU6, Aokap9 The expression cassette of the gene target sequence, sgRNA and Aspergillus oryzae U6 terminator TU6 was inserted into the gene editing vector pPRTII-Cas9 to generate the gene editing vector pPRTII-Cas9-Aokap9, and Aspergillus oryzae was subjected to protoplast genetic transformation to screen strains with high kojic acid production, thereby increasing the kojic acid production of Aspergillus oryzae.

3. A kind of Aspergillus oryzae according to claim 2 Aokap9 The application of genes is characterized in that Aspergillus oryzae Aokap9 Aspergillus oryzae Aokap9 The amino acid sequence of the gene is SEQ ID No.2:MAPSHLLCTFSPSRLTLFLSPPIFLLSSYLVICLSFAFRVYTFNSNTLVQIKTHLNNNTSTFIMSTTTTSVAASATASCNSVTYWQLPVDDAACALPKTGNYSDVMDK CCSPAKVTDFDNGCGLYCLAQGQSVGDLKDCLRKNGAQDGREFYCKGNDTATATASAPSSTKTGDKTGTATGSGASSTSSDSAAYAIQPAVSKGGLGLLAMVFCSALMGVVA.

4. A kind of Aspergillus oryzae according to claim 3 Aokap9 The application of genes is characterized in that Inhibit Aspergillus oryzae Aokap9 The method for screening strains with high kojic acid production is to obtain the expression or activity of the gene by overlapping PCR technology. Aokap9 The target gRNA expression cassette PU6-Aokap9-sgRNA-TU6 is then placed Aokap9 The target gRNA expression box PU6-Aokap9-sgRNA-TU6 is connected to the gene editing vector pPRTII-Cas9 through recombination to obtain the gene editing vector pPTRII-Cas9-Aokap9, and the gene editing vector pPTRII-Cas9-Aokap9 is used to perform protoplast genetic transformation on Aspergillus oryzae to increase the kojic acid production of Aspergillus oryzae.

5. A kind of Aspergillus oryzae according to claim 4 Aokap9 The application of genes is characterized in that In the overlapping PCR technique, the gene editing vector pPRTII-Cas9-kojA is used as a template, and primers PU6-F and PU6-Aokap9-R are used for amplification to obtain a Aokap9 The target PU6 fragment PU6-Aokap9; in addition, the gene editing vector pPRTII-Cas9-kojA was used as a template, and primers TU6-Aokap9-F and TU6-R were used for amplification to obtain a fragment containing Aokap9 The target gRNA and U6 terminator fragment Aokap9-sgRNA-TU6; Aokap9 The target fragment of PU6 is PU6-Aokap9 and contains Aokap9 The target gRNA and U6 terminator fragment Aokap9-sgRNA-TU6 were used as templates, and primers PU6-F and TU6-R were used for amplification to obtain a fragment containing Aokap9 sgRNA expression cassette for the target PU6-Aokap9-sgRNA-TU6.

6. A kind of Aspergillus oryzae according to claim 5 Aokap9 The application of genes is characterized in that The primers PU6-F and PU6- Aokap9 -R, Primer TU6- Aokap9 The sequences of primers TU6-F and TU6-R are as follows: PU6-F:CGACTCTAGAGGATCCCCGGGTAATGCCGGCTCATTCAAA; PU6-Aokap9-R:ATCTTTCTCCTGTCAAGCTAACTTGTTCTTCTTTACAATGATTTATTTA; TU6-Aokap9-F:TAGCTTGACAGGAGAAAGATGTTTTAGAGCTAGAAATAGCAAGTTAAA; TU6-R:AATTCGAGCTCGGTACCCGGGAGCAGCTCTATATCACGTGACG。

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