Aspergillus niger recombinant bacterium AnCT-XynB-K-agdA and application thereof

Abstract

The invention discloses an aspergillus niger recombinant bacterium AnCT-XynB-K-agdA and application thereof, and belongs to the technical field of microbiology and bioengineering. According to the invention, Aspergillus niger AnCat is taken as an expression host, firstly, an acid protease regulatory factor prtT is knocked out to obtain a defective strain, and then different chaperonins are subjected to fusion expression by optimizing integration sites of a laccase lcc9 expression cassette, so that the high-expression laccase chaperonin is obtained. According to the method, an expression vector pC3-5 'agdA-PcitA-XynB-L-lcc9-hph-3' agdA is constructed, a recombinant bacterium AnCT-XynB-K-agdA is further obtained, the enzyme activity of the extracellular laccase subjected to shake flask fermentation reaches 1821.2 U / L, and finally, the recombinant efficient expression of the laccase Lcc9 in aspergillus niger is realized.

Description

Technical Field

[0001] This invention relates to a recombinant Aspergillus niger strain AnCT-XynB-K-agdA and its applications, belonging to the fields of microbiology and bioengineering technology. Background Technology

[0002] Aspergillus niger has a wide range of growth temperature and pH conditions, and can grow in the range of 6-47℃ and pH 1.4-9.8. Due to its vigorous protein secretion and strong reproductive capacity, Aspergillus niger has been gradually developed into a multi-purpose cell factory for more than a century, and is widely used in industrial fermentation to produce various products and as a host bacterium for the production of recombinant proteins. In addition, Aspergillus niger has good safety and has been certified as a GRAS-rated safe strain by the U.S. Food and Drug Administration (FDA). Expression systems based on Aspergillus niger as a chassis host have the following advantages: (1) good safety and can be used in the food industry; (2) well-developed protein post-translational modification mechanism, suitable for expressing eukaryotic proteins; (3) can grow rapidly in low-cost fermentation feedstocks, suitable for high-density fermentation.

[0003] Extracellular degradation by proteases is one of the bottlenecks limiting the efficient expression of target proteins in *Aspergillus niger*. PepA is the most abundant protease in *Aspergillus niger*, accounting for 80-85% of total protease activity. In addition, pepsinoids such as PepAa, PeAb, PepAc, and PepAd also significantly degrade recombinantly expressed proteins. Therefore, constructing protease-deficient *Aspergillus niger* strains is an effective way to increase the expression level of target proteins. Researchers increased the expression level of laccase from *Trametes versicolor* by 21%, 42%, and 30% respectively by knocking out pepAa, pepAb, and pepAd in *A. niger* GICC2773. PrtT, a protease regulator in *Aspergillus niger*, significantly downregulates the activity of extracellular proteases by knocking out prtT. Therefore, regulating the activity of extracellular proteases in *Aspergillus niger* by knocking out prtT is particularly important for improving the expression level of target proteins.

[0004] Currently, exogenous genes in Aspergillus niger expression systems are typically expressed through genomic integration, i.e., by using integration plasmids to integrate the target gene's coding frame onto the chromosome. This integration can be categorized into site-directed integration and random integration. The integration site of the target gene also significantly impacts protein expression. Because the integration of the target gene into the host genome specifically disrupts relevant sites, and different sites affect the transcriptional level of the target gene, it can produce some potential unknown effects. Therefore, when exogenous proteins are expressed through site-directed integration at different gene loci in the host genome, the extracellular expression levels of the recombinant proteins may vary significantly. Thus, selecting a suitable integration site for the expression of the target protein is crucial for the expression of heterologous proteins.

[0005] Fusion proteins are proteins created by linking two or more genes encoding proteins with different biological functions together, resulting in the expression of bifunctional / multifunctional proteins. This artificial linking of the coding regions of two or more genes end-to-end, with expression controlled by the same regulatory sequence within the same organism, allows host cells to effectively recognize the protein sequence fused to the C-terminus of the target protein, thus reducing the degradation of the foreign protein by the host system to some extent. Furthermore, fusion expression requires the selection of a suitable linker sequence. Its selection has a significant impact on protein folding and stability. Currently, commonly used linker peptides for constructing fusion proteins include flexible linker peptides (GGGGS)n (n<6), α-helical linker peptides (EAAAK)n (n<6), and rigid linker peptides (XP)n. Another linker peptide, PTANVISKRAPV, can be disrupted by the extracellular acidic protease KexB secreted by the bacterial strain, thereby allowing the fused proteins to separate.

[0006] Laccases are copper-containing polyphenol oxidases that catalyze the oxidation of phenolic and non-phenolic compounds. Due to their broad substrate range and water-only product, they are considered "green" biocatalysts with significant application value. Laccases are widely distributed in plants, fungi, insects, bacteria, lichens, and sponges, but fungi and bacteria remain the primary sources, with over 80% of currently discovered laccases originating from fungi. Compared to bacterial laccases, fungal laccases have advantages such as higher redox potential, higher specific activity, and a broader substrate range. However, the long fermentation preparation cycle and low expression levels of fungal laccases limit their application in some industrial fields. Furthermore, fungal laccases exhibit optimal activity under acidic conditions, with activity essentially zero at pH > 7. Therefore, discovering fungal laccases with highly efficient catalytic activity under moderately alkaline conditions and achieving their efficient preparation is crucial for expanding the applications of laccases. Summary of the Invention

[0007] This invention provides a recombinant Aspergillus niger strain AnCT-XynB-K-agdA and its applications. The recombinant Aspergillus niger strain of this invention uses Aspergillus niger (AnCat) as the expression host. By knocking out the regulatory factor prtT of the acidic protease, a prtT-deficient strain of AnCat is obtained. Then, by optimizing the integration site of laccase LCC9 and optimizing different fusion expressions, pC3-5'agdA-P is achieved. citA Using XynB-L-lcc9-hph-3'agdA as the expression vector, recombinant bacteria AnCT-XynB-K-agdA were obtained. After shake-flask fermentation for 4-7 days, the shake-flask fermentation enzyme activity of laccase lcc9 reached 1821.2 U / L, which is of great significance for the industrial application of laccase lcc9.

[0008] The aforementioned Aspergillus niger AnCat was deposited at the China Center for Type Culture Collection (CCTCC) on January 16, 2025, under the classification name Aspergillus niger MA70.15-PcatSPCat, accession number CCTCC NO: M 2025169, and deposit address: Wuhan University, Wuhan, China.

[0009] The amino acid sequence of the laccase lcc9 is shown in SEQ ID NO:1.

[0010] The original nucleotide sequence encoding laccase lcc9 in the genome of the gray-capped Coprinus comatus is shown in SEQ ID NO:2.

[0011] The recombinant Aspergillus niger strain of this invention, AnCT-XynB-K-agdA, classified as Aspergillus nigerMA70.15-AnRTXynB-K-agdA-hph, has been deposited at the China Center for Type Culture Collection (CCTCC) on September 29, 2025, with accession number CCTCC NO: M20252148.

[0012] This invention relates to a recombinant Aspergillus niger strain, AnCT-XynB-K-agdA, which has the extracellular acidic protease regulator prtT knocked out. Its nucleotide sequence is shown in SEQ ID NO:3. The construction method includes the following steps:

[0013] Step 1: The prtT knockout vector was constructed based on the pFC330 vector. The prtT sgRNA promoter and terminator are derived from the U6 promoter of Aspergillus oryzae, and the nucleotide sequence of the prtT sgRNA is shown in SEQ ID NO:4.

[0014] Step 2: The constructed pFC330-prtT knockout vector was transformed into protoplasts prepared from Aspergillus niger (AnCat). The transformation method was based on polyethylene glycol (PEG)-mediated protoplast transformation, which included mixing protoplast suspension, recombinant plasmid and PEG buffer solution in an ice bath, then adding PEG buffer solution, osmotic stabilizer and soft agar medium, mixing and transferring to regeneration medium to obtain transformants. Transformants were picked and screened on screening plates to successfully obtain the prtT-deficient strain of AnCat.

[0015] Step 3: Using pC3 as the vector backbone, and employing P with a nucleotide sequence as shown in SEQ ID NO:5 citA The promoter, SP shown in SEQ ID NO:6 GlaA Signal peptides were used to construct different expression vectors, and the expression cassette of laccase LCC9 was integrated into 10 different sites through site-specific integration, with the optimal integration site being the agdA site.

[0016] Step 4: Using the expression vector pC3-5'agdA-P citA -SP Gla Using -lcc9-hph-3'agdA as the vector backbone, the nucleotide sequences of xylanase, truncated catalase, and truncated glucosylase are fused before laccase lcc9, as shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively, to construct different fusion expression vectors.

[0017] Step 5: Transform different fusion expression vectors into protoplasts prepared from the prtT-deficient strain of Aspergillus niger (AnCat).

[0018] The genetic transformation method is as follows: 150-200 μL of protoplast suspension is aspirated into a centrifuge tube, recombinant plasmid (4-10 μg) and PEG buffer solution are added, and the mixture is incubated on ice for 15-40 min, then PEG buffer solution is added and the mixture is placed at room temperature for 5-30 min.

[0019] The protoplast regeneration method is as follows: after mixing the transformation solution, osmotic stabilizer and hypertonic soft agar medium, the mixture is transferred to the regeneration medium and cultured upright at 28 °C for 4 to 7 days to obtain the transformed strain.

[0020] The screening method for transformants is as follows: transformants are picked and placed onto screening plates for screening. The components (w / v) involved in the screening plates include: glucose 1.5-3%, NaNO3 0.2-0.4%, KCl 0.1-0.3%, MgSO4·7H2O 0.04-0.06%, KH2PO4 0.05-0.15%, FeSO4·7H2O 0.001%, agar powder 1.5-2%, and hygromycin B 600-800 ng / L.

[0021] The solid plate used for activation culture is a PDA solid plate, and 1 L of culture medium contains the following components: 150-250 g of potato, 15-25 g of glucose, and 15-20 g of agar.

[0022] The liquid culture medium is YPD medium, and 1 L of the medium contains the following components: 15-25 g of peptone, 5-15 g of yeast extract, and 15-25 g of glucose.

[0023] The osmotic pressure stabilizer component comprises: CaCl2 40-60 mM, sorbitol 1.0-1.4 mM, Tris-HCl 8-12 mM, pH 7.5.

[0024] The PEG buffer solution comprises: PEG 4000 40-80% (w / v), CaCl2 40-60 mM, Tris-HCl 5-15 mM, pH 7.5.

[0025] The hypertonic soft agar medium composition (w / v) is as follows: sucrose 30-35%, NaNO3 0.2-0.4%, KCl 0.1-0.3%, MgSO4·7H2O 0.02-0.07%, KH2PO4 0.1-0.2%, FeSO4·7H2O 0.001-0.003%, and agar powder 1%.

[0026] The regeneration culture medium components are the same as those of hypertonic soft agar culture medium, except that the amount of agar powder added (w / v) is 1.5-2%.

[0027] The screening medium components (w / v) comprise: glucose 1.5-3%, NaNO3 0.2-0.4%, KCl 0.1-0.3% (w / v), MgSO4·7H2O 0.04-0.06%, KH2PO4 0.05-0.15%, FeSO4·7H2O 0.001%, agar powder 1.5-2%, and hygromycin B 600-800 ng / L.

[0028] The shake-flask fermentation medium components (w / v) comprise: 50-80 mM NaNO3, 5-10 mM KCl, 100-300 mM K2HPO4, 1-5 mM MgSO4, 1%-2% glucose (w / v), 0.1%-2% CaCO3, 0.05-0.2 M glycine, 2-5 mM CuSO4, and 0.1% trace elements (70-80 mM ZnSO4, 20-30 mM MnCl2, 15-20 mM FeSO4, 6-8 mM CoCl2, 6-8 mM CuSO4, 6-8 mM Na2MoO4, and 150-200 mM EDTA). The pH of the fermentation medium is adjusted to 5.5 using citric acid. The 0.1% in the trace element content refers to 0.1 mL of trace element solution added to 100 mL of medium.

[0029] This invention also provides the application of the above-mentioned recombinant Aspergillus niger AnCT-XynB-K-agdA in the efficient preparation of laccase Lcc9 from Coprinus gracilis.

[0030] The specific steps include:

[0031] The recombinant Aspergillus niger strain was spread on a PDA solid plate and cultured for 3-5 days until it had fully sporulated. Then, a physiological saline solution containing 0.05% Tween 80 was added, and spores were scraped off to prepare a spore suspension. The fresh spore suspension was inoculated into a fermentation medium (1×10⁻⁶). 5 The fermentation broth was incubated at 30℃ and 200 rpm for 7 days (spores / mL). 1 mL of the fermentation broth was taken daily, centrifuged at 12000×g for 5 min, and the supernatant was stored at 4℃. The fermentation supernatant was used to determine laccase activity and expression.

[0032] This invention successfully achieved efficient recombinant expression of laccase Lcc9 in Aspergillus niger. Using the technical solution of this invention, Aspergillus niger (AnCat) was used as the expression host. By knocking out the regulatory factor prtT of the acidic protease, a prtT-deficient strain of AnCat was obtained. Then, by optimizing the integration site of laccase Lcc9 and different fusion expressions, pC3-5'agdA-P was successfully expressed. citA Using XynB-L-lcc9-hph-3'agdA as the expression vector, recombinant bacteria AnCT-XynB-K-agdA were obtained. After shake-flask fermentation for 4-7 days, the shake-flask fermentation enzyme activity of laccase lcc9 reached 1821.2 U / L, which is of great significance for the industrial application of laccase lcc9. Attached Figure Description

[0033] Figure 1The prtT knockout vector pFC330-prtT constructed in this invention is shown in the diagram. A represents the plasmid map; B represents colony PCR identification; C represents plasmid restriction enzyme digestion verification; and D represents the restriction enzyme digestion verification results of the successfully knocked-out *Aspergillus niger* strain. 1-3 represent transformant 1, transformant 2, and transformant 3, respectively.

[0034] Figure 2 In Figure A, the data on laccase activity of the prtT-deficient strain of AnCat constructed in this invention during shake-flask fermentation are shown; in Figure B, the data on the Native-PAGE detection of the supernatant from shake-flask fermentation of the prtT-deficient recombinant Aspergillus niger constructed in AnCat are shown.

[0035] Figure 3 The results are for the verification of laccase expression vectors with different integration sites constructed in this invention; where A is: plasmid map of laccase expression vectors with different integration sites, B is colony PCR identification, M1 is DNA Maker, and 1~2 are transformant 1 and transformant 2 respectively; C is plasmid restriction enzyme digestion verification, M2 is DNA Maker, and 1~2 are transformant 1 and transformant 2 respectively.

[0036] Figure 4 This invention presents the results of shake-flask fermentation of recombinant Aspergillus niger with different integration sites to produce laccase Lcc9. A represents the data on shake-flask fermentation of laccase Lcc9 from recombinant Aspergillus niger with different integration sites; B represents the Native-PAGE analysis of the supernatant from shake-flask fermentation of recombinant Aspergillus niger with the laccase Lcc9 expression cassette integrated into the agdA site.

[0037] Figure 5 The results are for the verification of different fusion expression recombinant vectors of the present invention; where M1 is DNA Marker, A is colony PCR identification, 1~3 are transformant 1, transformant 2, and transformant 3 respectively; B is plasmid enzyme digestion verification, M2 is DNA Marker, 1~2 are transformant 1 and transformant 2 respectively.

[0038] Figure 6 This invention presents the results of shake-flask fermentation of recombinant Aspergillus niger with different fusion expressions to produce laccase Lcc9. A represents the data on shake-flask fermentation of laccase Lcc9 produced by recombinant Aspergillus niger with different fusion expressions; B represents the Native-PAGE analysis of the shake-flask fermentation supernatant of recombinant Aspergillus niger AnCT-XynB-K-agdA. Detailed Implementation

[0039] Unless otherwise specified, the implementation methods in the following embodiments are all conventional methods.

[0040] In one embodiment of the present invention, the Aspergillus niger AnCat has the accession number CCTCC M 2025169.

[0041] pUC57-prtT-sgRNA and pUC57-hph were synthesized by Shanghai Sangon Biotech.

[0042] The culture media and solutions involved in the following examples are as follows:

[0043] PDA solid medium (1 L): 200 g potato, 20 g glucose, 15-20 g agar, with an additional 10 mM uracil as needed.

[0044] YPD medium (w / v): 2% peptone, 1% yeast extract, 2% glucose, with an additional 10mM uracil as needed.

[0045] Hypertonic solid CD medium (1 L): sucrose 34% (w / v), NaNO3 0.3% (w / v), KCl 0.2% (w / v), MgSO4·7H2O 0.05% (w / v), KH2PO4 0.1% (w / v), FeSO4·7H2O 0.001% (w / v), agar powder 1.5-2% (w / v).

[0046] Hyperosmolar CD soft agar medium (w / v): sucrose 34%, NaNO3 0.3%, KCl 0.2%, MgSO4·7H2O 0.05%, KH2PO4 0.1%, FeSO4·7H2O 0.001%, agar powder 1%.

[0047] Screening medium (w / v): glucose 2%, NaNO3 0.3%, KCl 0.2%, MgSO4·7H2O 0.05%, KH2PO4 0.1%, FeSO4·7H2O 0.001%, agar powder 1.5-2%, and hygromycin B 600-800 ng / L

[0048] STC buffer: Anhydrous CaCl2 50 mM, Sorbitol 1.2 M, Tris-HCl 10 mM, pH 7.5.

[0049] PEG buffer: PEG 4000 60% (w / v), CaCl2 50 mM, Tris-HCl 10 mM, pH 7.5.

[0050] Fermentation medium: 10.1%-2% CaCO3, 0.05-0.2 M glycine, 2-5 mM CuSO4 4、50-80 mM NaNO3, 5-10 mM KCl, 100-300 mM K2HPO4, 1-5 mM MgSO4, 1%-2% glucose (w / v), 0.1% trace elements (70-80 mM ZnSO4, 20-30 mM MnCl2, 15-20 mM FeSO4, 6-8 mM CoCl2, 6-8 mM CuSO4, 6-8 mM Na2MoO4, and 150-200 mM EDTA).

[0051] The detection methods involved in the following embodiments are as follows:

[0052] Laccase activity: Laccase activity was determined using the ABTS method. Specifically, 950 μL of sodium tartrate buffer (100 mmol / L, pH 4.0) was mixed with 33 μL of 15 mmol / L ABTS and 17 μL of appropriately diluted enzyme solution. The mixture was thoroughly mixed and then incubated in a water bath at 30°C for 3 min. The reaction was terminated by incubating on ice for 30 s. The absorbance of the reaction solution was measured at a wavelength of λ = 420 nm.

[0053] Formula for calculating laccase activity: E (U / L) = OD 420 ×Dilution factor×555.56.

[0054] Laccase expression: Laccase expression in the fermentation broth was detected by native-pAGE. Specific procedures: The supernatant of the fermentation broth, diluted to a certain factor, was mixed with 2× loading buffer, and 10 μL was loaded into the wells of the active gel. Electrophoresis was performed in a VE-180A miniature vertical electrophoresis tank at 80 V for 30 min, followed by electrophoresis at 120 V for 3 h. The active gel was then removed and incubated at room temperature in 100 mmol / L pH 4.0 sodium tartrate buffer (0.5 mmol / L LABTS).

[0055] Example 1: Effect of knockout of the acidic protease regulator prtT on laccase expression

[0056] The knockout vector pFC330-prtT used in this invention is constructed based on the pFC330 vector, which is disclosed in SCN, BJN, EMK, et al. A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi.[J]. PLOS ONE, 2015, 10(7):e0133085. The knockout vector pFC330-prtT is based on the pFC330 vector, recombined with the sgRNA of prtT and the repair template. A schematic diagram of the vector structure of the knockout vector pFC330-prtT is shown below. Figure 1 .

[0057] The specific steps are as follows:

[0058] Fragments of prtT-sgRNA and the upstream and downstream homologous arms of the repair template were obtained, and the upstream and downstream homologous arms were ligated by fusion PCR. Homologous recombination ligation was performed with the linearized pFC330 vector using the CloneExpress II One-step Cloning Kit from Novizumi. The pFC330 vector containing prtT-linked sgRNA was then linearized and ligated with the fusion fragment of the upstream and downstream homologous arms for homologous recombination.

[0059] The nucleotide sequence of prtT's sgRNA is shown in SEQ ID NO:4. It was obtained by PCR using primer F1 / R1 with the synthetic gene pUC57-prtT-sgRNA as a template. The sequences of primers F1 / R1 are shown in Table 1. The linearized pFC330 vector was obtained by PCR using primer F2 / R2 with plasmid pFC330 as a template. Primers F2 / R2 are shown in Table 1. Homologous arms were obtained by PCR using Aspergillus niger (AnCat) hyphae as a template using primers F3 / R3 and F4 / R4, and then fused using fusion PCR. Primers F3 / R3 and F4 / R4 are shown in Table 1. The linearized vector was obtained by PCR using primer F5 / R5 with the pFC330 plasmid containing prtT's sgRNA as a template. Primers F5 / R5 are shown in Table 1.

[0060]

[0061] The fusion PCR used consisted of two steps. The first step involved recombination and fusion using homologous sequences between the upstream and downstream homologous arms without primers; the fusion PCR system is shown in Table 2. The second step involved large-scale PCR amplification of the recombination product from the first step using primers F3 / R4. Linearization of the vector pFC330 was achieved by inverse PCR; the sequences of primers F2 / R2 are shown in Table 1. Linearization of the pFC330 plasmid ligated with prtT sgRNA was also achieved by inverse PCR; the sequences of primers F5 / R5 are shown in Table 1.

[0062]

[0063] PCR conditions: 94℃ pre-denaturation, 5 min; 98℃ denaturation, 30 s, 55℃ annealing, 15 s, 72℃ extension, 1 min, 15 cycles; 72℃, 10 min. PCR products were recovered by gel extraction.

[0064] The connection system between the fusion fragment and the linearized vector pFC330 is shown in Table 3.

[0065]

[0066] Connection conditions: React at 37℃ for 30 min.

[0067] Depend on Figure 1 -B and 1-C indicate that colony PCR, enzyme digestion verification, and sequencing have confirmed the successful construction of the prtT knockout vector pFC330-prtT.

[0068] Using *Aspergillus niger* (AnCat) as the host strain, protoplasts were prepared, and the constructed vector pFC330-prtT was transformed into them. The specific process is as follows:

[0069] (1) Preparation of protoplasts

[0070] A. Activation culture of the strain

[0071] The Aspergillus niger AnCat glycerol tube preservative stored at -80℃ was appropriately diluted, spread onto PDA solid medium, and incubated at 28℃ for 3-5 days.

[0072] B. Culture of mycelium (liquid culture method)

[0073] Take an activated plate covered with spores, cut off a piece of mycelium and inoculate it into YPD liquid medium, and incubate at 28-32℃ and 180-220 rpm for 48 h.

[0074] C. Collection of mycelium

[0075] Filter the bacterial cells using a Buchner funnel lined with double-layered filter paper until the cells are dry. Then wash the cells twice with sterile deionized water and twice with NaCl solution, filtering again until the cells are dry. Scrape off the cells with a clean spatula and collect them in an Erlenmeyer flask or EP tube, then weigh them.

[0076] D. Enzymatic hydrolysis of mycelium

[0077] Add the enzymatic hydrolysate at a ratio of mycelium to enzymatic hydrolysate of 1:10 (w / v), and place it in a water bath shaker at 37℃ and 100 rpm for 3 h for enzymatic hydrolysis.

[0078] The enzymatic hydrolysate consisted of: 2% (w / v) cellulase, 1% (w / v) lysozyme, 0.5% (w / v) lysozyme, 1% (w / v) snailase, 0.8 M NaCl, and 10% (v / v) 100 mM pH 6.0 sodium phosphate buffer.

[0079] E. Collection of protoplasts

[0080] Filter the enzyme digest using lens paper (smooth side) to collect the protoplasts. Then rinse the test tubes containing the enzyme digest and the lens paper with NaCl. Centrifuge at 3000-4000 rpm for 10 min and discard the supernatant.

[0081] F. Purification of protoplasts

[0082] Add STC buffer (osmotic stabilizer), gently aspirate with a de-pointed pipette tip to resuspend the protoplasts, and centrifuge at 3000-4000 rpm for 10 min at 4°C to collect the protoplasts. Repeat this step once. Add an appropriate amount of STC buffer (osmotic stabilizer), gently aspirate with a de-pointed pipette tip to resuspend the protoplasts, and obtain a protoplast suspension.

[0083] (2) Transformation of protoplasts

[0084] Pipette 150-200 μL of protoplast suspension into 2 mL centrifuge tubes, then add 20 μL of knockout vector pFC330-prtT (4-10 μg) and 50 μL of PEG buffer solution. Gently mix by inverting the tubes, repeating this process every 10 min. After incubating on ice for 30 min, add 1.5 mL of PEG buffer solution and incubate at room temperature for 25 min.

[0085] (3) Regeneration of protoplasts

[0086] Add 2.5 mL of STC buffer and 5 mL of hypertonic CD soft agar medium to a 10 mL centrifuge tube, mix well, transfer the protoplast transformation mixture to a 10 mL centrifuge tube, mix with a pre-cooled 5 mL de-pointed pipette tip, and then pipette 5 mL onto two hypertonic solid CD medium plates. Gently rotate the plate to spread the soft agar evenly on the plate. Incubate at 28°C, upright, for 4-7 days.

[0087] (4) Screening and identification of transformants

[0088] Twenty transformants from the regeneration plate were transferred to a screening plate. After culturing for 2-3 days, mycelia were scraped for mycelial PCR verification. The success of prtT gene knockout was determined by the presence of the BamHI restriction site added to the repair template.

[0089] The results showed that the prtT knockout strain of AnCat was successfully obtained (e.g. Figure 1 -D)

[0090] (5) Shake-flask fermentation of recombinant Aspergillus niger

[0091] The obtained AnCat prtT knockout strain was spread on PDA solid plates and cultured for 3-5 days until it had fully sporulated. Then, a physiological saline solution containing 0.05% Tween 80 was added, and spores were scraped off to prepare a spore suspension. The fresh spore suspension was inoculated into fermentation medium (1×10⁻⁶). 5 (spores / mL), cultured at 30℃ and 200 rpm for 7 days. 1 mL of fermentation broth sample was taken daily, centrifuged at 12000×g for 5 min, and the supernatant was stored at 4℃. The fermentation supernatant was used to determine laccase activity and expression.

[0092] Laccase expression: After mixing the fermentation broth supernatant with 2× loading buffer, 10 μL was loaded into the wells of the active gel and electrophoresed at 80 V for 30 min in a VE-180A miniature vertical electrophoresis tank, followed by electrophoresis at 120 V for 3 h. The active gel was then removed and incubated at room temperature in 100 mmol / L pH 4.0 sodium tartrate buffer (0.5 mmol / L ABTS).

[0093] The results showed that the laccase activity of the prtT-deficient strain of AnCat reached its peak on day 5, with an activity of 1225.5 U / L, which was 1.21 times that of strain AnCat (1004.5 U / L). Figure 2 -A). Figure 2 -B is a Native-PAGE image of the shake-flask fermentation supernatant of the prtT-deficient strain, indicating that knocking out the acidic protease regulator prtT is effective in increasing the expression level of laccase Lcc9.

[0094] Example 2: Construction of expression vectors containing laccase derived from *Coprinus gracilis* integrated at different sites

[0095] The expression vector used in this invention uses pC3-C2 as its backbone. The pC3-C2 vector is disclosed in Yao D, Liu X, Wang H, et al. Enhanced extracellular production of Coprinopsiscinerea laccase Lcc9 in Aspergillus niger by gene expression cassette and bioprocess optimization[J]. BMC Biotechnology, 2024, 24(1):95-95. The expression vector is obtained by replacing the upstream and downstream homologous arms of the pC3-C2 vector. A schematic diagram of the vector structure is shown below. Figure 3 -A.

[0096] The specific steps are as follows:

[0097] (1) Obtaining long fragments by fusion

[0098] Upstream and downstream homologous arms of target genes aamA, amyA, agdA, pepA, pepB, pepN, prtT, goxC, oahA, and cpyS, as well as the expression cassette of lcc9 and hygromycin B, were obtained. The four fragments were then combined into a single long fragment by fusion PCR. Homologous recombination was performed between the long fragment and the linearized pC3-C2 fragment using the CloneExpress II One-step Cloning Kit from Novizan.

[0099] The expression cassette of lcc9 was obtained by PCR amplification using primer F6 / R6 with pC3-C2 as a template. Primers are shown in Table 4. Other primers used included F7 / R7, F8 / R8, F9 / R9, F10 / R10, F11 / R11, F12 / R12, F13 / R13, F14 / R14, F15 / R15, F16 / R16, F17 / R17, F18 / R18, and F19 / R19. Using *Aspergillus niger* (AnCat) hyphae as templates, primers F20 / R20, F21 / R21, F22 / R22, F23 / R23, F24 / R24, F25 / R25, and F26 / R26 were used to amplify the upstream and downstream homologous arms of the target genes aamA, amyA, agdA, pepA, pepB, pepN, prtT, oxC, oahA, and cpyS by PCR. Primers are shown in Table 4. Using primer F27 / R27 as a template with pUC57-hph, the hygromycin B hph fragment was amplified by PCR. Primers are shown in Table 4. The nucleotide sequence of hph is shown in SEQ ID NO:10. Using primer F28 / R28 as a template with pC3-C2, a linearized vector was obtained by PCR. Primers are shown in Table 4.

[0100]

[0101]

[0102] (2) Obtaining expression vectors for laccase LCC9 integration at different sites

[0103] The fusion PCR used consisted of two steps. The first step involved recombination and fusion of homologous sequences between elements without primers; the fusion PCR system is shown in Table 5. The second step used primers F7 / R27, F9 / R27, F11 / R27, F13 / R27, F15 / R27, F17 / R27, F19 / R27, F21 / R27, F23 / R27, and F25 / R27 to amplify the recombination products from the first step by PCR. Linearization of the vector pC3 was achieved by inverse PCR; the sequences of primers F5 / R5 are shown in Table 1.

[0104]

[0105] PCR conditions: 94℃ pre-denaturation, 5 min; 98℃ denaturation, 30 s; 55℃ annealing, 15 s; 72℃ extension, 2 min 36 s, 15 cycles; 72℃, 10 min. PCR products were recovered by gel electrophoresis.

[0106] The connection system between the fusion fragment and the linearized vector pC3-C2 is shown in Table 6.

[0107]

[0108] Connection conditions: React at 37℃ for 30 min.

[0109] Depend on Figure 3 It can be seen that the expression vectors pC3-aamA, pC3-amyA, pC3-agdA, pC3-pepA, pC3-pepB, pC3-pepN, pC3-prtT, pC3-goxC, pC3-oahA, and pC3-cpyS have been successfully constructed through colony PCR, enzyme digestion verification, and sequencing.

[0110] Example 3: Effects of laccase LCC9 integration at different sites on its expression

[0111] Using a prtT-deficient strain of Aspergillus niger (AnCat) as the host strain, protoplasts were prepared, and the vector constructed above was transferred into them. Specifically, the preparation, transformation, and regeneration of protoplasts were carried out according to Example 1.

[0112] Transformed strains from the regeneration plates were transferred to hygromycin resistance selection plates and cultured for 2-3 days before mycelial PCR verification. Recombinant Aspergillus niger strains AnCT-aamA, AnCT-amyA, AnCT-agdA, AnCT-pepA, AnCT-pepB, AnCT-pepN, AnCT-prtT, AnCT-goxC, AnCT-oahA, and AnCT-cpyS were finally prepared.

[0113] The obtained recombinant Aspergillus niger strain was spread on a PDA solid plate and cultured for 3-5 days until it had fully conjugated. Then, a physiological saline solution containing 0.05% Tween 80 was added, and spores were scraped off to prepare a spore suspension. The fresh spore suspension was inoculated into a fermentation medium (1×10⁻⁶). 5 The fermentation broth was incubated at 30℃ and 200 rpm for 7 days (spores / mL). 1 mL of the fermentation broth was taken daily, centrifuged at 12000×g for 5 min, and the supernatant was stored at 4℃. The fermentation supernatant was used to determine laccase activity and expression.

[0114] The results showed that the laccase activity of strain AnCT-agdA reached its peak on day 5, with an activity of 1597.5 U / L, which was 1.6 times that of strain AnCat (1004.5 U / L). Figure 4 -A). Figure 4 -B is a gel image of the activity of the supernatant from the shake-flask fermentation of strain AnCT-agdA.

[0115] Example 4: Construction of different fusion expression vectors containing laccase derived from Coprinus macrocephala.

[0116] The expression vector used in this invention uses pC3-C2 as its backbone, and different fusion proteins are added to the pC3-C2 vector. See Table 7 for details.

[0117]

[0118] Fragments XynB-K, XynB-G, XynB-R, Cat-K, Cat-G, Cat-R, Gla 1-G, and Gla 2-G were amplified using *Aspergillus niger* (AnCat) hyphae as templates via primers F29 / R29, F29 / R30, F29 / R31, F30 / R32, F30 / R33, F30 / R34, F31 / R35, and F31 / R36, respectively. Primers are listed in Table 8. A linearized vector was obtained using the pC3-agdA vector as a template via primer F37 / R37. Primers are listed in Table 8. Homologous recombination ligation of each fragment with the linearized pC3-agdA vector was performed using the Novozymes CloneExpress II One-step Cloning Kit. The one-step cloning ligation system is listed in Table 9.

[0119]

[0120]

[0121] Connection conditions: React at 37℃ for 30 min.

[0122] Depend on Figure 5 It can be seen that the expression vectors pC3-XynB-K-agdA, pC3-XynB-G-agdA, pC3-XynB-R-agdA, pC3-Cat-K-agdA, pC3-Cat-G-agdA, pC3-Cat-R-agdA, pC3-Gla1-G, and pC3-Gla2-G have been successfully constructed through colony PCR, enzyme digestion verification, and sequencing.

[0123] Example 5: Effects of different fusion expressions on laccase expression

[0124] Using a prtT-deficient strain of Aspergillus niger (AnCat) as the host strain, protoplasts were prepared, and the vector constructed above was transferred into them. Specifically, the preparation, transformation, and regeneration of protoplasts were carried out according to Example 1.

[0125] Transformed strains from the regeneration plates were transferred to hygromycin resistance selection plates and cultured for 2-3 days before mycelial PCR verification. Recombinant Aspergillus niger strains AnCT-XynB-K-agdA, AnCT-XynB-R-agdA, AnCT-XynB-G-agdA, AnCT-Cat-L-agdA, AnCT-Cat-G-agdA, AnCT-Cat-R-agdA, AnCT-Gla1-G-agdA, and AnCT-Gla2-G-agdA were finally prepared.

[0126] The obtained recombinant Aspergillus niger strain was spread on a PDA solid plate and cultured for 3-5 days until it had fully conjugated. Then, a physiological saline solution containing 0.05% Tween 80 was added, and spores were scraped off to prepare a spore suspension. The fresh spore suspension was inoculated into a fermentation medium (1×10⁻⁶). 5 The fermentation broth was incubated at 30℃ and 200 rpm for 7 days (spores / mL). 1 mL of the fermentation broth was taken daily, centrifuged at 12000×g for 5 min, and the supernatant was stored at 4℃. The fermentation supernatant was used to determine laccase activity and expression.

[0127] The results showed that the laccase activity of strain AnCT-XynB-K-agdA reached its peak on day 5, with an activity of 1821.2 U / L, which was 1.81 times that of strain AnCat (1004.5 U / L). Figure 6 -A). Figure 6 -B is a gel image of the activity of the supernatant from the shake-flask fermentation of strain AnCT-XynB-K-agdA.

[0128] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims. SEQ ID NO:1

[0129] MRLLTLTLLFLRIVSASIGPTADLHIVNNNIQPDGFTRSAVLAGTSSTVGTFPGPLISGAKGTTFFMNVVNHLTDTTMLTTTSIHWHGMFQHRTSWADGPIGVTQCPITPGDSFLYQFSVPDQAGTFWYHSHHSAQYCDGLRGPLVIYDKLDPNRALYDIDDETTVITLADWYHTPSPQLNSFPTPDATLINGKGRYSGGPAAPLSNITVVQGKRYRFRLVSMSCLPNYVFSIDGHTMNIIEVDGISVTPTTVDSIQIFAGQRYSFVLTASQPTDNYWIRALPNVGTPGFNGGINSAILQYAGAPNQDPTSTQTASVMPMRETDLHPLVNPGAPGPAKPGAADININLNIVFNYTLFKYTLNGAPFDPPTVPVLLQILSGAKTAQEMLPPGSVYVLPRDKVVEVTMPAVALAIGGPHPLHLHGNTFDVIRSAGSSTYNFVNPVRRDVVSIGQNGDNVTIRFTTNNAGPWFLHCHIDWHMVLGLGVVFAVDVPTISSTNPPAAWNQLCPTYNGQSAP SEQ ID NO:2

[0130] SEQ ID NO:3

[0131] SEQ ID NO:4

[0132] GCAGGCGGTTGCAAGCGATCTATCGTGAATTTGTGGAGGGACGTAATGGTGAAATGCCGAATGGCGAAGCAGGTCGGGAGATCGGTGGTCGGACGAGCCTGATGTAGATGGGTGCATATCTTTCTTTTCTCTTTCACAATTTCAAGATCATGTATTAGAACTACATTGCTATGTTTCAGCAATATATATGCATAGCTAGAGAGCGATGTTACGATGAGACATGTAATCTTTGAACATTTCTCTGTACAGCTTGTCAGAGTTTCGAAGGTTAAGCTCGAGAGCAAGGAGGGGAAAAGAAGACAAAAAAGACACTCGGT CCGGTCTCGAACCGACGACCTTTCGCACACACGTAGAAGGTACAGAAACTCAACACCTCCAAAAGAGGTCTGTAAGAATGAAAGCATGAGTACCTGACAACCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGCTTTTTTTTTGAGCATTTATCAGCTTGATATAGAGGTAGGAATGTATGGAGGTGCAGAATGGCTATTTTGTTATTGGAGCGGGTTCGAAACGGAGGGCAGGAGACTTTTTCTAAATACGTCACGTGATATAGAGCTGCT SEQ ID NO:5

[0133] AACAATGAAAAAAAAATTCACTTGACCATGTAGAGGTATGAACTTATATGTCGAGCAATGGGTGTAAAGTATTACCACTACGTACTTGTATTGTTGTTATGGCTGGGGAAATCCGCGCCCATGGAAACTCAATGGGTGGGCAAAATTGTGGATGAGATGGCGACTGCCGACGTCACTAGCCATGGACCAACAGCTCTACTGTGGCACTCCGTTTACGGATGAGACGGCGCCGATGATCGTCACAATAATAGTAGCCCGAGGCGGCATTACAGTGACTATATCTATATTCCAGCACATGCATGAATGACCGTTTGATAGCAAGTGACAGCTCTGGTCTTTGCTTTTCAACAGATCCCTATAAGCTCGAGCAAGTATGAGAGAATGACATGGTAGAACTCTAAAAGGTAGTGATAAATACATAGCGTAAAAAGGACACACAGACCAAGAAACAAAACGCAAGGAAAGAGAGGATTTTACTCCGGACTCCGTTCCGTACAACGGAATCAAGCGGGTAAGGGTCACTACCAAAACATGATGGATCCTTCTGATTGCCGCTGGAAAGCGCGCCGAAATCCTGGCTCAGACTCGGGATATCGGCCAGGTTGAGACTAGTGTGACCGTAAGATACAGCCGCCCACCATTACCTTGCGGCGTTGCCGTGTGTTACCGTACAGTCCGCGTACCGGCTGTATTGGTACGGCCAGATGGAAAAAAAGGGCCGCGCAAAAGAGCTTCTCCATAACTTGGGAACACCGTGCGGCCCATCTTCCTTTTTCTTTTCTCTACCGTGTCCTGATATACCCTCTTTCTCTCCTCTCCTTTTCTTCTTCCAACCACTCAACTTACTGCAAGTTGTTCACATTCGCC SEQ ID NO:6

[0134] ATGTCCTTCCGCTCCCTGCTGGCCCTGTCCGGCCTGGTCTGCACCGGCCTGGCC SEQ ID NO:7

[0135] ATGCTCACCAAGAACCTTCTCCTCTGCTTTGCCGCGGCTAAGGCTGCTCTGGCTGTTCCCCACGACTCTGTGGCCCAGCGTTCGGATGCCTTGCACATGCTCTCTGAGCGCTCGACCCCGAGCTCGACCGGCGAGAACAACGGCTTCTACTACTCCTTCTGGACCGACGGCGGTGGAGACGTGACCTACACCAACGGAGATGCTGGTGCCTACACTGTTGAGTGGTCCAACGTGGGCAACTTTGTCGGTGGAAAGGGCTGGAACCCCGGAAGTGCGCAGTAAGTTAATCCTCCTCACGCTATCTCTTTGCAGTAACCAATGAAACGATTACTCACAAAATAATCAGGGACATCACCTACAGCGGCACCTTCACCCCTAGCGGCAACGGCTACCTCTCCGTCTATGGCTGGACCACTGACCCTCTGATCGAGTACTACATCGTCGAGTCCTACGGCGACTACAACCCCGGCAGTGGAGGCACGTACAAGGGCACCGTCACCTCGGACGGATCCGTTTACGATATCTACACGGCTACCCGTACCAATGCTGCTTCCATTCAGGGAACCGCTACCTTCACTCAGTACTGGTCCGTTCGCCAGAACAAGAGAGTTGGCGGAACCGTTACCACCTCCAACCACTTCAATGCTTGGGCTAAGCTGGGAATGAACCTGGGTACTCACAACTACCAGATCGTGGCTACCGAGGGTTACCAGAGCAGTGGATCTTCGTCCATCACTGTTCAGTAA SEQ ID NO:8

[0136] ATGCGTCCCATCGGTCTCGTTGGCCTCATTGGCCTTGCCAATGCCGCCTGCCCTTACATGAGTCGTGACCTCTCTGCTCGCGACTCCTCAGATGCCTCAGAATCCACTGAGGAATTCCTCTCTCAGTACTACCTCAATGACAATAATACTTACCTCACTTCCGACGTGGGCGGTCCCATTGAGGACCAGAACAGTTTGAGCGCTGGTGAACGCGGCCCCACTCTGCTAGAGGACTTTATCTTCCGCCAGAAGATCCAGCGCTTCGATCATGAACGAGTAAGTCCTTTTGTTTATCTACTACGCAATTGCACCACTAAGAATGTAGGTTCCCGAACGCGCCGTACATGCCCGTGGCGTCGGTGCCCACGGTGTCTTCGTCTCCTACGGCGACTTCTCCAACCTCACCGCGGCCTCATTCCTCTCTGAACAGGGCAAGGAAACTCCCATGTTCGTGCGCTTCTCCACTGTCGCTGGTAGTCGTGGCAGTACCGATCTGGCGCGCGATGTGCACGGTTTTGCCACTCGCTTCTACACCGATGAGGGTAATTTCGGTAAGCTATCCCCTGGTTACCGTACTCTTAGCCAAACACTAACTCGTCAGATCTCGTGGGAAACAACATCCCCGTGTTCTTCATCCAGGATGCCATCCAGTTCCCCGACCTGATCCACGCCGTCAAGCCCAGAGGTGACAGTGAGATCCCGCAAGCCGCGACTGCTCACGACTCGGCCTGGGACTTCTTCAGCCAGCAGCCTAGCGCTCTGCATACTCTATTCTGGGCAATGGCTGGACACGGTATCCCGCGCTCCTTCCGGCATGTTGACGGCTTTGGCGTGCACACATACCGGCTGGTGACGGACAGCGGCGCGTCGAAGCTGGTCAAGTTCCATTGGAAGTCGCTGCAGGGCAAGGCCAGTATGGTCTGGGAAGAGGCGCAACAAGTCTCGGGCAAGAACTCGGACTTTATGCGCCAGGACCTGTGGACATCGATTGAGTATGGAC。 SEQ ID NO:9

[0137] ATGTCGTTCCGATCTCTACTCGCCCTGAGCGGCCTCGTCTGCACAGGGTTGGCAAATGTGATTTCCAAGCGCGCGACCTTGGATTCATGGTTGAGCAACGAAGCGACCGTGGCTCGTACTGCCATCCTGAATAACATCGGGGCGGACGGTGCTTGGGTGTCGGGCGCGGACTCTGGCATTGTCGTTGCTAGTCCCAGCACGGATAACCCGGACTGTATGTTTCGAGCTCAGATTTAGTATGAGTGTGTCATTGATTGATTGATGCTGACTGGCGTGTCGTTTGTTGTAGACTTCTACACCTGGACTCGCGACTCTGGTCTCGTCCTCAAGACCCTCGTCGATCTCTTCCGAAATGGAGATACCAGTCTCCTCTCCACCATTGAGAACTACATCTCCGCCCAGGCAATTGTCCAGGGTATCAGTAACCCCTCTGGTGATCTGTCCAGCGGCGCTGGTCTCGGTGAACCCAAGTTCAATGTCGATGAGACTGCCTACACTGGTTCTTGGGGACGGCCGCAGCGAGATGGTCCGGCTCTGAGAGCAACTGCTATGATCGGCTTCGGGCAGTGGCTGCTTGTATGTTCTCCACCCCCTTGCGTCTGATCTGTGACATATGTAGCTGACTGGTCAGGACAATGGCTACACCAGCACCGCAACGGACATTGTTTGGCCCCTCGTTAGGAACGACCTGTCGTATGTGGCTCAATACTGGAACCAGACAGGATATGGTGTGTTTGTTTTATTTTAAATTTCCAAAGATGCGCCAGCAGAGCTAACCCGCGATCGCAGATCTCTGGGAA。

Claims

1. A recombinant Aspergillus niger strain AnCT-XynB-K-agdA, characterized in that: The recombinant Aspergillus niger strain uses Aspergillus niger (AnCat) as the expression host. By knocking out the regulatory factor prtT of the acidic protease, an AnCat prtT-deficient strain was obtained. Then, by optimizing the integration site of laccase LCC9 and different fusion expressions, pC3-5'agdA-P was used. citA Using -XynB-L-lcc9-hph-3'agdA as the expression vector, the recombinant Aspergillus niger strain AnCT-XynB-K-agdA was obtained.

2. The recombinant Aspergillus niger strain AnCT-XynB-K-agdA according to claim 1, characterized in that: The recombinant Aspergillus niger strain AnCT-XynB-K-agdA, classified as Aspergillus niger MA70.15-AnRTXynB-K-agdA-hph, has been deposited at the China Center for Type Culture Collection (CCTCC) on September 29, 2025, with accession number CCTCC NO: M20252148.

3. The application of the recombinant Aspergillus niger strain AnCT-XynB-K-agdA as described in claim 1 or 2 in the efficient preparation of laccase Lcc9 from Coprinus grayis.

4. The application according to claim 3, characterized in that: The amino acid sequence of the laccase lcc9 is shown in SEQ ID NO:1.

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