A gene-edited strain of Aspergillus niger for oxalic acid production, and a construction method and application thereof

Abstract

The present invention relates to a method for constructing a gene-edited strain of Aspergillus niger, and belongs to the field of bioengineering technology. The Aspergillus niger gene-edited strain is: using Aspergillus niger as a starting strain, overexpressing the oxaloacetate acetylhydrolase gene oahA, knocking out the citric acid transporter gene citT, or simultaneously overexpressing the oxaloacetate acetylhydrolase gene oahA and knocking out the citric acid transporter gene citT, to obtain the Aspergillus niger gene-edited strain. The present invention also provides methods for constructing, screening, and subculturing the edited strain, as well as a scheme for fermenting and producing oxalic acid and quantitatively analyzing organic acid products. Compared with the prior art, the Aspergillus niger edited strain of the present invention has strong vitality and strong acid production ability, and oxalic acid is dominant in the organic acid produced by fermentation, which provides a theoretical model and technical support for the fermentation production of oxalic acid, fungal organic acid metabolism regulation, and biometallurgy related research.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a gene-edited Aspergillus niger strain for oxalic acid production, a construction method and an application thereof. Background Art

[0002] Aspergillus niger is a saprophytic fungus of the genus Aspergillus, belonging to the subphylum Ascomycota. It has a wide distribution range, is easy to cultivate, can adapt to a variety of environments, and has strong stress resistance. The main organic acids produced by Aspergillus niger include citric acid, oxalic acid, gluconic acid, etc. Oxalic acid is a dibasic organic acid composed of carbon, hydrogen and oxygen atoms with strong acidity and chelating ability. Oxalic acid secreted by microorganisms can be used in wet bioleaching metallurgy. oahA is a gene encoding oxaloacetate acetylhydrolase in Aspergillus niger, which is a key enzyme in the only pathway of oxalic acid synthesis in Aspergillus niger. citT is a gene encoding a citrate transporter in Aspergillus niger, which can secrete citric acid in the cytoplasm out of the cell.

[0003] Biometallurgy is a method of smelting metal ores using microorganisms and their secretions to enrich and recover target metals. It is often used to treat low-grade ores. There are many types of microorganisms that can be used for biometallurgy, including bacteria such as Acidithiobacillus ferrooxidans and silicate bacteria, and fungi such as Aspergillus niger and Penicillium.

[0004] At present, the bioleaching process is limited to Acidithiobacillus and sulfide ores, and the bioleaching of oxide ores is difficult. Although silicate bacteria with oxalic acid as the main leaching agent can dissolve some oxide ores, they are difficult to culture; although Aspergillus niger is an excellent oxalic acid fermentation production strain, the oxalic acid production is poor in the acidic leaching environment. In addition, since organic acid metabolism is strictly restricted by central carbon regulation, the current research on the organic acid metabolism regulation mode of fungi is relatively limited and lacks effective models. Summary of the invention

[0005] The purpose of the present invention is to solve the problems existing in the prior art and provide a gene-edited Aspergillus niger strain and a construction method thereof to achieve continuous and efficient production of oxalic acid.

[0006] Technical Solution

[0007] A method for constructing an Aspergillus niger gene-edited strain: using Aspergillus niger as a starting strain, overexpressing the oxaloacetate acetylhydrolase gene oahA, knocking out the citric acid transporter gene citT, or simultaneously overexpressing the oxaloacetate acetylhydrolase gene oahA and knocking out the citric acid transporter gene citT, to obtain the Aspergillus niger gene-edited strain.

[0008] Furthermore, the starting strain is Aspergillus niger CBS 513.88.

[0009] Furthermore, the sequence of the oxaloacetate acetylhydrolase gene oahA is shown in SEQ ID NO:1.

[0010] Furthermore, the sequence of the citric acid transporter gene citT is shown in SEQ ID NO:2.

[0011] The Aspergillus niger gene-edited strain was constructed using the above method.

[0012] Application of the Aspergillus niger gene-edited strain constructed by the above method in fermentation production of oxalic acid.

[0013] Beneficial effects of the present invention: Compared with the prior art, the present invention has the following advantages:

[0014] (1) Compared with the wild strain Aspergillus niger CBS 513.88, the edited strain has a stronger ability to produce acid;

[0015] (2) Oxalic acid dominated the organic acids produced by fermentation of the citT knockout bacteria;

[0016] (3) Under low pH conditions, the edited strain can also maintain a good oxalic acid production efficiency, and has great application potential for oxalic acid bioleaching of low-grade oxide ores;

[0017] (4) The phenotypes of the edited strains of oahA and citT were significantly different, and they have research value as metabolic modification models. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The colony morphology of the three Aspergillus niger gene-edited strains and the wild-type Aspergillus niger obtained in Example 1 on a plate containing bromocresol green;

[0019] Figure 2 The bacterial morphology of the three Aspergillus niger gene-edited strains and the wild-type Aspergillus niger obtained in Example 1 in liquid shake flask culture;

[0020] Figure 3 The gene sequencing results of the three Aspergillus niger gene-edited strains obtained in Example 1;

[0021] Figure 4 is the standard curve of oxalic acid content;

[0022] Figure 5 These are the test results of the contents of oxalic acid and citric acid in the fermentation broths of the three Aspergillus niger edited strains and the Aspergillus niger wild strain obtained in Example 1. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention are clearly and completely described below in conjunction with the accompanying drawings. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention.

[0024] In the following examples, the pUC57 plasmid, pFC332 vector, pUC57-PGA5-agdA upstream and downstream homology arm plasmid, KpnI enzyme, NheI enzyme, XhoI enzyme, BglⅡ enzyme, and PacⅠ enzyme used were all purchased from Beijing Anbiqi Biotechnology Co., Ltd., but are not limited thereto.

[0025] Example 1

[0026] Construction of A. niger editing strain:

[0027] 1. Construction of oahA overexpression fragment

[0028] The oahA gene sequence was optimized according to the codon bias of the Aspergillus niger gene (the sequence is shown in SEQ ID NO: 1), and a KpnI restriction site was added to the 5' end of the target gene oahA, and an NheI restriction site was added to the 3' end. The oahA gene fragment was obtained by digesting the pUC57-oahA plasmid with KpnI and NheI, and connected to the pUC57-PGA5-agdA upstream and downstream homology arm plasmid digested with the same enzymes to construct the pUC57-oahA-agdA upstream and downstream homology arm plasmid. The above plasmid was digested with XhoI and PacⅠ to obtain an oahA overexpression cassette fragment containing the upstream and downstream homology arms of agdA, which was used for site-specific integration of the oahA gene. This fragment contains the upstream and downstream homology arms of agdA, a promoter, a terminator and the oahA gene.

[0029] 2. Construction of CRISPR / Cas9-based citT knockout plasmid

[0030] According to the citT gene (sequence as shown in SEQ ID NO: 2), the target primers sgRNA-citTF and sgRNA-citTR were designed, and the primers were annealed to form double strands and connected to the intermediate plasmid pUC57 to form the plasmid pUC57-sgRNA-citT containing the target sgRNA expression cassette. The plasmid was digested with BglⅡ and PacⅠ to obtain a linear sgRNA-target expression cassette fragment, which was ligated to the pFC332 vector after the same digestion to construct the pFC332-sgRNA-citT targeted knockout plasmid.

[0031] 3. Primer Design

[0032] Vector: pFC332, Aspergillus niger strain: Aspergillus niger CBS 513.88 (hereinafter referred to as CBS513.88WT), purchased from Beijing Anbiqi Biotechnology Co., Ltd., primer design was performed, and the relevant primers and sequences are shown in Table 1:

[0033] Table 1

[0034]

[0035] In Table 1, all primer synthesis and sequencing work were completed by Beijing Anbiqi Biotechnology Co., Ltd.

[0036] 4. Protoplast transformation, transformant screening and sequencing

[0037] Aspergillus niger edited strain CBS 513.88OE: Using Aspergillus niger CBS 513.88 as the starting strain, the strain was obtained by overexpressing the oxaloacetate acetylhydrolase gene oahA through the CRISPR-Cas9 system. Specific method: The oahA overexpression fragment constructed above was transferred into Aspergillus niger through the protoplast transformation method, and the transformants were cultured on MM+200μg / mL hygromycin plates at 30℃ for 5-7 days. The transformants were picked and subcultured on PDA+200μg / mL hygromycin plates for 2-3 generations. An appropriate amount of mycelium was picked and placed in a 1.5mL centrifuge tube to extract the genome, and the targeted gene was amplified by PCR using verification primers. After preliminary verification by agarose gel electrophoresis, it was delivered to BGI for sequencing verification.

[0038] Aspergillus niger edited strain CBS 513.88KO: Aspergillus niger CBS 513.88 was used as the starting strain, and the strain was obtained by knocking out the citrate transporter gene citT through the CRISPR-Cas9 system. The specific method was as follows: The pFC332-sgRNA-citT targeted knockout plasmid constructed above was transferred into Aspergillus niger through the protoplast transformation method, and the transformants were cultured on MM+200μg / mL hygromycin plates at 30°C for 5-7 days. The transformants were picked and cultured on PDA+200μg / mL hygromycin plates for 2-3 generations. An appropriate amount of mycelium was picked and the genome was extracted in a 1.5mL centrifuge tube. The targeted gene was amplified by PCR using verification primers. After preliminary verification by agarose gel electrophoresis, it was delivered to BGI for sequencing verification.

[0039] Aspergillus niger edited strain CBS 513.88DE: Aspergillus niger CBS 513.88 was used as the starting strain, and the strain was obtained by simultaneously overexpressing the oxaloacetate acetylhydrolase gene oahA and knocking out the citrate transporter gene citT through the CRISPR-Cas9 system. The specific method was as follows: the pFC332-sgRNA-citT targeted knockout plasmid and the oahA overexpression fragment constructed above were transferred into Aspergillus niger by protoplast transformation. The transformants were cultured on MM+200μg / mL hygromycin plates at 30℃ for 5-7 days, and the transformants were picked and subcultured on PDA+200μg / mL hygromycin plates for 2-3 generations. An appropriate amount of hyphae was picked and the genome was extracted in a 1.5mL centrifuge tube. The targeted gene was amplified by PCR using the verification primers. After preliminary verification by agarose gel electrophoresis, it was delivered to BGI for sequencing verification.

[0040] MM culture medium formula: glucose 0.5%, ammonium nitrate 1g / L, potassium dihydrogen phosphate 0.5g / L, disodium hydrogen phosphate 1.5g / L, sodium chloride 1g / L, magnesium sulfate heptahydrate 0.2g / L, pH 7.2.

[0041] PDA plate culture medium formula: potato extract powder 5g / L, glucose 20g / L, agar 20g / L.

[0042] Figure 1 The colony morphology of the three Aspergillus niger gene-edited strains and the wild-type Aspergillus niger obtained in Example 1 on the plate containing bromocresol green, wherein WT is the wild-type Aspergillus niger CBS 513.88, KO is the edited strain CBS 513.88KO, OE is the edited strain CBS 513.88OE, and DE is the edited strain CBS 513.88DE. Figure 1 It can be seen that the diameters of the color circles of the wild type WT and the edited strain DE are comparable, the diameter of the color circle of the edited strain KO is smaller, and the diameter of the color circle of the edited strain OE is larger.

[0043] Figure 2 The bacterial morphology of the three Aspergillus niger gene-edited strains and the wild-type Aspergillus niger obtained in Example 1 in liquid shake flask culture, wherein WT is the wild-type CBS 513.88, KO is the edited strain CBS 513.88KO, OE is the edited strain CBS513.88OE, and DE is the edited strain CBS 513.88DE. Figure 2 It can be seen that the wild bacteria WT, the edited strain OE and the edited strain DE all formed mycelial balls, but the edited strain KO could not form mycelial balls. The bacteria appeared as a scattered and uniform suspension in the culture medium. The edited strain DE formed mycelial balls with more small diameters and secreted more yellow metabolites into the culture medium.

[0044] The gene sequencing results of the three Aspergillus niger gene-edited strains obtained in Example 1 are as follows: Figure 3 ,in, Figure 3 (A) is the sequencing results of the edited strains KO and DE. Figure 3 (B) is the sequencing result of the edited strain OE. Figure 3 It can be seen that the citT gene sequence of the edited strain KO and the edited strain DE has base deletions resulting in frameshift mutations, causing the gene to lose its function; at the same time, the sequences of the new copies of oahA integrated into the edited strains OE and DE are correct, indicating that the oahA gene is correctly integrated into the genomes of the Aspergillus niger strains OE and DE.

[0045] The three gene-edited engineered bacteria obtained in the example were named CBS 513.88OE, CBS 513.88KO, and CBS 513.88DE, respectively. The strains Aspergillus niger CBS 513.88, CBS 513.88OE, CBS 513.88KO and CBS 513.88DE were deposited in China Center for Type Culture Collection, and the deposit information is as follows: Aspergillus niger CBS 513.88, deposited in China Center for Type Culture Collection, with the deposit number CCTCC No: M20232377, and the deposit date is November 29, 2023; Aspergillus niger CBS 513.88OE, with the deposit number CCTCC No: M20232380, and the deposit date is November 29, 2023; Aspergillus niger CBS513.88KO, with the deposit number CCTCC No: M 20232378, the preservation date is November 29, 2023; Aspergillus niger CBS 513.88DE, the preservation number is CCTCC No: M 20232379, the preservation date is November 29, 2023.

[0046] Example 2

[0047] Oxalic acid fermentation culture of Aspergillus niger edited strain:

[0048] a. Activation culture of Aspergillus niger strains: The three Aspergillus niger edited strains (CBS 513.88OE, CBS 513.88KO, CBS 513.88DE) and the Aspergillus niger wild strain (CBS 513.88WT) of Example 1 were inoculated on PDA plates, respectively, and activated and cultured at 30°C for 6 days. The PDA composition was (g / L): potato extract powder: 5; glucose: 20; agar: 20.

[0049] b. Spore collection of Aspergillus niger strains: Scrape fresh spores with sterile dd H2O on a PDA plate full of bacteria to obtain a spore suspension. Dilute the spore suspension to a concentration of 10 7 Pieces / mL.

[0050] c Preparation of fermentation medium: 100 mL of liquid culture medium was placed in a 250 mL conical flask, and the initial pH of the liquid culture medium was adjusted to 5.5 with HCl and NaOH. The liquid culture medium was sterilized at 121°C for 15 minutes. The components of the liquid culture medium (g / L) were: sucrose: 50; sodium nitrate: 1.5; potassium dihydrogen phosphate: 0.5; anhydrous magnesium sulfate: 0.0122; anhydrous potassium chloride: 0.025; manganese sulfate pentahydrate: 0.014; and yeast extract: 1.6.

[0051] d Spore inoculation: Add the obtained spore suspension to the liquid culture medium, and the final spore concentration is 10 5 / mL, 30℃, 60r / min in a constant temperature shaking incubator for bioleaching.

[0052] Ion chromatography was used to detect the components and contents of organic matter in the fermentation broth.

[0053] Detection method: The fermentation broth was centrifuged to obtain a filtered solution and bacteria. The solution was filtered and pre-treated through a filter membrane with a pore size of 0.22 μm. The obtained solution was tested on an ion chromatograph (ICS-900, Dionex China Co., Ltd.). Chromatographic column: AS23 anion analysis column (250 mm × 4 mm, Thermo Fisher Scientific); guard column (50 mm × 4 mm); column temperature: 30 ° C; suppressor type: ASRS 300 × 4 mm, 50 mA, self-circulating suppression; conductivity detector; prepare eluent with a concentration of 4.5 mmol / L Na2CO3 and 0.8 mmol / L NaHCO3, isocratic elution; flow rate: 1.0 mL / min; injection volume: 25 μ L; single sample time: 30 min.

[0054] Different concentrations of oxalic acid standards (1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, 16 mg / mL) were used to draw the standard curve. The standard curve is as follows: Figure 4 shown.

[0055] Figure 5The test results of the content of oxalic acid and citric acid in the fermentation broth of the three Aspergillus niger edited strains and the Aspergillus niger wild strain obtained in Example 1 show that the oxalic acid content in the fermentation broth of the four strains WT, DE, OE, and KO is 0.316 mg / mL, 4.25 mg / mL, 2.98 mg / mL, and 2.47 mg / mL, and the citric acid content is 2.98 mg / mL, 6.74 mg / mL, 0.0931 mg / mL, and 0.135 mg / mL. This shows that the three Aspergillus niger edited strains constructed in Example 1 of the present invention have strong vitality and are suitable for the fermentation production of oxalic acid, which provides a theoretical model and technical support for the fermentation production of oxalic acid, the regulation of fungal organic acid metabolism, and biometallurgy related research.

[0056] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing a gene-edited strain of Aspergillus niger, characterized in that: Using Aspergillus niger as the starting strain, the oxaloacetate acetylhydrolase gene was overexpressed oah and knockout of citrate transporter gene citT , the Aspergillus niger gene-edited strain obtained; the starting strain is Aspergillus niger Aspergillus niger CBS 513.88; the oxaloacetate acetylhydrolase gene oah The sequence is shown in SEQ ID NO: 1; the citrate transporter gene citT The sequence is shown in SEQ ID NO:

2.

2. The gene-edited Aspergillus niger strain constructed by the method of claim 1.

3. Use of the Aspergillus niger gene-edited strain constructed by the method of claim 1 in the fermentation production of oxalic acid.

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