Aspergillus niger, methods of making and using same, and methods of producing citric acid and / or succinic acid

Abstract

The application relates to the field of genetic engineering, and discloses an Aspergillus niger, a construction method and application of the Aspergillus niger, and a method for producing citric acid and / or succinic acid. Aspergillus niger The Aspergillus niger is obtained by simultaneously overexpressing alpha-amylase and aspartic protease in a starting strain. The Aspergillus niger provided by the application can realize efficient degradation and conversion of starch by synergistically overexpressing amyA genes and pepA genes, solve the technical bottleneck that the original strain is limited in growth in a starch-based fermentation system and has insufficient acid production capacity, can efficiently produce succinic acid and citric acid by taking low-cost starch as the only carbon source, significantly reduce the industrial production cost of organic acid, and promote the green and sustainable upgrading of the fermentation industry.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, specifically to a strain of Aspergillus niger (… Aspergillus niger ) and its construction methods and applications, and methods for producing citric acid and / or succinic acid. Background Technology

[0002] Succinic acid and citric acid are indispensable core raw materials in the food, pharmaceutical, and chemical industries, and are widely used in food preservation, drug synthesis, and industrial catalysis, with huge market demand. Currently, the mainstream method for industrial production of these organic acids is microbial fermentation, among which Aspergillus niger has become one of the most commonly used production strains due to its low nutritional requirements, strong enzyme production capacity, and high biosafety.

[0003] However, existing fermentation processes face key technological bottlenecks: traditional Aspergillus niger strains (such as Aspergillus niger ATCC 1015) naturally lack the ability to efficiently degrade starch, requiring glucose as the primary carbon source in industrial production. However, glucose is a high-cost raw material and relies on grain processing, resulting in high organic acid production costs, severely restricting the industry's large-scale development and market competitiveness. Although starches (corn starch, potato starch, etc.) are widely available and inexpensive, making them ideal alternative carbon sources, common Aspergillus niger has extremely low starch degradation efficiency, preventing its direct use as a fermentation feedstock for efficient acid production and limiting its application in industrial production.

[0004] To address these issues, the industry has attempted to modify Aspergillus niger strains using genetic engineering techniques, with the core idea being the introduction of starch degradation-related genes. However, existing technologies mostly employ single-gene overexpression strategies, and the starch degradation capacity of the modified strains still does not meet the requirements for industrial production, while also exhibiting problems such as low gene expression efficiency and poor fermentation stability.

[0005] Meanwhile, as a core regulatory element of gene expression, the promoter's strength directly determines the transcription efficiency and protein synthesis amount of the target gene. In existing modification schemes, the promoter selection is not well-suited to the target gene, resulting in limited expression levels of starch-degrading enzymes and making it difficult to achieve rapid and complete starch degradation. Furthermore, traditional strain modification methods suffer from low transformation efficiency and poor genetic stability, failing to meet the stringent performance requirements of industrial production.

[0006] Furthermore, in starch-based fermentation systems, starch tends to form a semi-solid viscous system after high-temperature sterilization. This leads to poor mycelial dispersion and low mass transfer efficiency of wild-type Aspergillus niger ATCC 1015 during shake-flask fermentation, further exacerbating the difficulty of starch utilization and severely restricting the growth, metabolism, and organic acid synthesis capabilities of the strain.

[0007] Therefore, by using genetic engineering to perform targeted modification of Aspergillus niger to achieve efficient synergistic expression of the amyA and pepA genes, thereby enhancing the starch degradation capacity of the strain and solving the problems of limited growth and low acid production efficiency of wild strains in starch-based fermentation systems, it is of great significance for promoting the use of low-cost starch to replace glucose as a fermentation carbon source in the organic acid industry. Summary of the Invention

[0008] The purpose of this invention is to overcome the problems of low starch utilization efficiency of Aspergillus niger strains, semi-solid starch after sterilization making shake-flask fermentation difficult, and the reliance on high-cost glucose carbon sources in traditional organic acid production. This invention provides a strain of Aspergillus niger, its construction method and application, and a method for producing citric acid and / or succinic acid. Aspergillus niger This technology enables efficient degradation and conversion of starch, solving the technical bottleneck of limited growth and insufficient acid production capacity of the original strain in starch-based fermentation systems. It can efficiently produce succinic acid and citric acid using low-cost starch as the sole carbon source, significantly reducing the industrial production cost of organic acids and promoting the green and sustainable upgrading of the fermentation industry.

[0009] To achieve the above objectives, the present invention provides, on the one hand, a strain of Aspergillus niger (… Aspergillus niger The Aspergillus niger was obtained by simultaneously overexpressing α-amylase and aspartic protease in the starting strain.

[0010] The second aspect of the present invention provides a *Aspergillus niger* ( Aspergillus niger The method for constructing a starting strain includes overexpressing the encoding gene for α-amylase and the encoding gene for aspartic protease in the starting strain.

[0011] The third aspect of the present invention provides Aspergillus niger as described above ( Aspergillus niger ) and / or Aspergillus niger constructed as described above ( Aspergillus niger Application in the degradation of starch to produce citric acid and / or succinic acid.

[0012] A fourth aspect of the present invention provides a method for producing citric acid and / or succinic acid, the method comprising: processing Aspergillus niger as described above (… Aspergillus niger ) and / or Aspergillus niger constructed as described above ( Aspergillus niger The fermentation culture is carried out; the carbon source for the fermentation culture is starch, and the amount of starch used is 70-110 g relative to 1 L of the fermentation culture medium used for the fermentation culture.

[0013] Through the above technical solution, the Aspergillus niger (Aspergillus) provided by the present invention... Aspergillus nigerBy synergistically overexpressing the amyA and pepA genes, the efficient degradation and conversion of starch is achieved, solving the technical bottleneck of limited growth and insufficient acid production capacity of the starting strain in the starch-based fermentation system. It can efficiently produce succinic acid and citric acid with low-cost starch as the sole carbon source, significantly reducing the industrial production cost of organic acids and promoting the green and sustainable upgrading of the fermentation industry. Attached Figure Description

[0014] Figure 1 It is the recombinant strain Aspergillus niger in Example 4 ( Aspergillus niger Figure 1 shows the results of reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative polymerase chain reaction (qPCR) detection of gene expression levels; the left figure is a semi-quantitative RT-PCR figure, and the right figure is a real-time quantitative PCR and qPCR figure. Figure 2 This is a comparison of the growth phenotypes of different strains in Example 4 on MM solid medium with glucose and starch as the sole carbon sources. Figure 3 This is a schematic diagram illustrating the starch degradation capacity of different strains in a starch-based fermentation medium, as shown in Example 4. Figure 4 The graph shows the results of acid production performance of different strains (excluding ATCC 1015) in fermentation medium with starch as the carbon source in Example 4, and the results of acid production performance of ATCC 1015 in fermentation medium with glucose as the carbon source. Detailed Implementation

[0015] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0016] The first aspect of this invention provides a strain of Aspergillus niger ( Aspergillus niger The Aspergillus niger was obtained by simultaneously overexpressing α-amylase and aspartic protease in the starting strain.

[0017] During the experimental process of the invention, the sterilized starch was in a semi-solid state. The inventors accidentally discovered that the starting strain was difficult to disperse evenly during shake-flask fermentation, while strains expressing the amyA and pepA genes dispersed well in the shake flask. The *Aspergillus niger* (*Aspergillus niger*) provided by this invention... Aspergillus nigerBy synergistically overexpressing the amyA and pepA genes, the efficient degradation and conversion of starch was achieved, overcoming the technical difficulties of limited growth and poor acid production capacity of the original strain in starch-based fermentation systems. It can utilize low-cost starch as the sole carbon source to efficiently produce succinic acid and citric acid, effectively reducing the cost of industrial production of organic acids and powerfully promoting the green, sustainable development and upgrading of the fermentation industry.

[0018] According to the present invention, preferably, the starting strain has the accession number ATCC 1015. This strain, as a model strain, has a clear genetic background, mature culture conditions, and basic acid-producing potential. Only targeted enhancement of starch utilization-related genes is needed to quickly achieve performance breakthroughs. Compared to screening unknown wild-type strains, this significantly shortens the research and development cycle and reduces costs associated with strain modification. The inventors discovered in their research that using this as a starting strain for modification can maximally preserve its original acid-producing metabolic pathways, reduce the growth and metabolic burden caused by heterologous modification, and lay the foundation for subsequent efficient organic acid production.

[0019] According to the present invention, preferably, the nucleotide sequence of the gene encoding the α-amylase is shown in SEQ ID NO. 1. The inventors discovered in their research that the α-amylase encoded by this sequence has a stronger affinity for starch molecules, can efficiently cleave the α-1,4-glycosidic bonds within starch, and rapidly degrades macromolecular starch into dextrins and oligosaccharides, providing a usable carbon source for the growth and metabolism of the strain. Compared with α-amylase genes from other sources, its heterologous expression efficiency in Aspergillus niger is higher and its enzyme activity is more stable.

[0020] According to the present invention, preferably, the nucleotide sequence of the gene encoding the aspartic protease is shown in SEQ ID NO. 2. The inventors discovered in their research that the aspartic protease encoded by this sequence not only assists in the breakdown of starch-binding proteins but also synergizes with α-amylase, further improving the overall hydrolysis efficiency of starch and solving the problem of incomplete starch degradation when only α-amylase is used. Simultaneously, this gene sequence is adapted to the codon bias of Aspergillus niger, and its expression level is significantly higher than that of non-optimized sequences.

[0021] According to the present invention, preferably, the Aspergillus niger ( Aspergillus nigerThe product contains promoter I for overexpressing the gene encoding α-amylase and promoter II for overexpressing the gene encoding aspartic protease; the genes encoding α-amylase and aspartic protease are provided in the form of a recombinant vector. The inventors discovered that dual promoters drive the expression of two target genes separately, avoiding the gene expression imbalance problem associated with single-promoter tandem expression, and achieving synchronous and efficient transcription of the amyA and pepA genes. Simultaneously, the combination with a strong promoter adapted to *Aspergillus niger* significantly enhances the transcription level of the target genes, thereby strengthening the strain's starch degradation capacity and acid production efficiency. Furthermore, the gene introduction method mediated by recombinant vectors enables stable integration and controllable expression of the target genes. Compared to breeding methods such as random mutation, gene modification is more targeted, precisely avoiding the negative impact of unrelated gene mutations on strain performance, and facilitating subsequent regulation and optimization of gene expression levels.

[0022] According to the present invention, preferably, promoter I is the Pgpd promoter; and promoter II is the Ptef promoter. The inventors discovered that both the Pgpd and Ptef promoters are strong constitutive promoters derived from *Aspergillus niger*, and their regulatory sequences are highly adapted to the transcriptional system of *Aspergillus niger*. Compared with heterologous promoters, they can significantly improve the transcriptional efficiency and expression stability of the target gene. Simultaneously, the two promoters drive the expression of the amyA and pepA genes respectively, effectively avoiding transcriptional interference and expression imbalance problems during tandem gene expression, achieving synergistic and efficient synthesis of α-amylase and aspartic protease, thereby maximizing the starch degradation capacity and acid production performance of the strain.

[0023] The second aspect of the present invention provides a *Aspergillus niger* ( Aspergillus niger A method for constructing Aspergillus niger (A. niger) is disclosed, comprising: overexpressing the encoding genes of α-amylase and aspartic protease in a starting strain. The inventors discovered in their research that this construction method, through targeted overexpression of the α-amylase encoding gene and the aspartic protease encoding gene, can precisely achieve the synergistic expression of the two functional proteins, significantly enhancing the strain's ability to degrade starch. This method has a clear operational pathway, strong reproducibility, and yields high-quality Aspergillus niger. Aspergillus niger It has stable genetic traits and can effectively solve the technical problem of wild strains being difficult to ferment in semi-solid starch culture medium.

[0024] The preferred embodiments of the method provided in the second aspect of the present invention refer to the preferred embodiments provided in the first aspect of the present invention, and will not be repeated hereby by the applicant.

[0025] The third aspect of the present invention provides Aspergillus niger as described above ( Aspergillus niger ) and / or Aspergillus niger constructed as described above ( Aspergillus nigerApplication of [a specific technology / method] in the degradation of starch to produce citric acid and / or succinic acid. This application can replace traditional high-cost glucose as a fermentation carbon source with low-cost starch, significantly reducing the industrial production cost of citric acid and succinic acid. This application can efficiently solve the fermentation problem caused by the semi-solid state after starch sterilization, realizing the fermentation of Aspergillus niger ([a specific type of starch]). Aspergillus niger Stable growth and efficient acid production in starch-based systems provide a new pathway for the green biomanufacturing of organic acids.

[0026] A fourth aspect of the present invention provides a method for producing citric acid and / or succinic acid, the method comprising: processing Aspergillus niger as described above (… Aspergillus niger ) and / or Aspergillus niger constructed as described above ( Aspergillus niger Fermentation is carried out using starch as the carbon source, with 70-110 g of starch used relative to 1 L of fermentation medium. The inventors discovered that this method uses low-cost starch as the sole fermentation carbon source, replacing the traditional high-cost glucose raw material, significantly reducing the industrial production cost of citric acid and succinic acid. This method relies on Aspergillus niger (… Aspergillus niger Its high-efficiency starch degradation capability solves the fermentation problem caused by the semi-solid state of starch after sterilization, and achieves stable and high production of organic acids, providing a feasible technical solution for green biomanufacturing.

[0027] According to the present invention, preferably, the fermentation conditions include: a time of 55-70 h, a temperature of 20-35℃, and a rotation speed of 180-220 rpm. The inventors discovered in their research that under these conditions, *Aspergillus niger* (a double-gene fungus)... Aspergillus niger It can efficiently and thoroughly degrade starch in fermentation medium, greatly improving degradation efficiency; at the same time, the optimized oscillation speed can ensure that the mycelium is evenly dispersed in the semi-solid starch system and ensure the dissolved oxygen content of the fermentation system, ultimately achieving high yield of succinic acid and citric acid.

[0028] The present invention will be described in detail below through examples. In the following examples, Aspergillus niger ATCC 1015 was obtained from the laboratory of Professor Huang He of Nanjing Normal University and was previously disclosed in patent CN202311000298.1; Aspergillus niger strain 513.88 was purchased from UNGAL GENETICS STOCK CENTER; and the remaining materials and reagents were all commercially available products.

[0029] The culture medium used in this invention and its preparation are as follows: PDA medium: 20% (w / v) potato broth, accurately weigh 200 g of peeled potatoes and cut them into approximately 1 cm pieces. 3Place small pieces of potato in a pot, add water, stir, and boil for about 30 minutes. After cooling, filter and squeeze through double-layered gauze to remove the potato puree. At the same time, add 20 g of glucose and 20 g of agar powder to the filtrate and stir until completely dissolved. Adjust the volume to 1 L, leaving the pH to set naturally without adjustment. Autoclave at 121℃ for 20 minutes.

[0030] MM medium (1L): 10 g glucose / starch, 1 ml Trace elements (trace element mixture), 50 ml 20×Salts (high concentration salt solution stock solution: 10.4 g / L KCl, 10.4 g / L MgSO4·7H2O, 30.4 g / L KH2PO4), 15 g agar.

[0031] Transformation medium (1L): 10 g glucose, 1 ml trace elements, 20×Salts (high-concentration salt solution stock solution: 10.4 g / L KCl, 10.4 g / L MgSO4·7H2O, 30.4 g / L KH2PO4), 1 g yeast extract, 218.6 g sorbitol, 1.7 g ammonium tartrate, 10 g agar, and add appropriate reagents as needed.

[0032] Trace elements (100 mL) Ingredients: 2.20 g ZnSO4·7H2O, 1.1 g H3BO3, 0.5 g MnCl2·4H2O, 0.16 g FeSO4·7H2O, 0.16 g CoCl2·5H2O, 0.16 g CuSO4·5H2O, 0.11g (NH4)6Mo7O 24 ·4H2O, 5 g Na4EDTA.

[0033] 20×Salts (1L): 10.4 g KCl, 10.4 g MgSO4·7H2O, 30.4 g KH2PO4.

[0034] Osmotic medium (1L): 144.48 g MgSO4, 0.8 g NaH2PO4, 0.5 g Na2HPO4 The formula for the Trapping buffer (trapping buffer used in protoplast preparation and purification) is as follows: Weigh 109.3 g of D-Sorbitol and 12.114 g of Tris, adjust the pH to 7 with HCl, add ultrapure water to a final volume of 1 L, and autoclave at 121°C for 20 min.

[0035] The STC buffer formulation is as follows: Weigh 218.6 g of D-Sorbitol (D-sorbitol is a key osmotic pressure stabilizer), 1.47 g of CaCl2·2H2O, and 1.2114 g of Tris. Adjust the pH to 7.5 with HCl, add ultrapure water to a final volume of 1 L, and autoclave at 121°C for 20 min.

[0036] The formulation of PEG buffer (whose core function is to promote the fusion of recombinant vectors and protoplasts and improve transformation efficiency) is as follows: Weigh 120 g of PEG4000, 1.47 g of CaCl2·2H2O, and 1.2114 g of Tris. Adjust the pH to 7.5 with HCl, add ultrapure water to a final volume of 200 mL, and autoclave at 121°C for 20 min.

[0037] The formula for KAc buffer (potassium acetate buffer) is as follows: Weigh 29.442 g of KAc, add 11.5 mL of HAc, and add ultrapure water to make up to 100 mL.

[0038] The DNA extraction buffer (lysis buffer) formula is as follows: NaCl 150 mM, Na2EDTA 60 mM, Tris-HCl 400 mM, SDS 1 g.

[0039] Seed culture medium preparation: 4% glucose by weight, 0.6% bacterial peptone by weight, 0.075% anhydrous potassium dihydrogen phosphate, 0.075% anhydrous dipotassium hydrogen phosphate, 0.01% magnesium sulfate heptahydrate, 0.01% calcium chloride dihydrate, and 0.0001-0.0005% trace elements (sodium chloride, ferrous sulfate heptahydrate, and anhydrous citric acid). Sterilize at 115℃ for 20 min.

[0040] The trace elements include sodium chloride 5 mg / L, ferrous sulfate heptahydrate 5 mg / L, and anhydrous citric acid 1 mg / L.

[0041] Fermentation medium preparation: 10 wt% starch, 0.6 wt% bacterial peptone, 0.015 wt% anhydrous potassium dihydrogen phosphate, 0.015 wt% anhydrous dipotassium hydrogen phosphate, 0.01 wt% calcium chloride dihydrate, 0.01 wt% magnesium sulfate heptahydrate, and 0.0001-0.0005 wt% trace elements (5 mg / L sodium chloride, 5 mg / L ferrous sulfate heptahydrate, and 1 mg / L anhydrous citric acid). Sterilize at 115℃ for 20 min. All percentages are final mass concentrations.

[0042] Example 1: Experimental Materials and Gene Cloning Experimental materials: 1. Starting strain: Aspergillus niger ATCC 1015.

[0043] 2. Gene source: Aspergillus niger strain 513.88 (which can secrete amyA α-amylase and pepA aspartic protease).

[0044] Gene cloning: Genes amyA (nucleotide sequence shown in SEQ ID NO. 1) and pepA (nucleotide sequence shown in SEQ ID NO. 2) were amplified from the genome of Aspergillus niger 513.88, cloned using the primers shown in Table 1, and sequenced for verification before use.

[0045] Table 1

[0046] Example 2: Carrier Construction The self-replicating Cas9 expression plasmid pFC330 (carrier number pyrG / hph selection marker) was purchased from Addgene.

[0047] In constructing the expression plasmids, a seamless cloning technique was employed (using Exnase II enzyme digestion, Cloning Express Super-One Cloning Kit V3, C117). The gpdA gene promoter I (Pgpd; its nucleotide sequence is shown in SEQ ID NO. 21) and the tefA gene promoter II (Ptef; its nucleotide sequence is shown in SEQ ID NO. 22) were inserted into the p-zero plasmid backbone containing the pyrG / hph selection marker, respectively. Two plasmids were ultimately obtained: pEASY-zero-pyrG-Pgpd and pEASY-zero-hph-Ptef.

[0048] To overexpress the amyA and pepA genes, the corresponding open reading frames (ORFs) were subcloned into the p-zero-pyrG-Pgpd vector to construct the recombinant vectors p-zero-pyrG-PgpdA-amyA and p-zero-pyrG-PgpdA-pepA.

[0049] In addition, the PtefA-pepA fragment was subcloned into the p-zero-hph vector to finally obtain the p-zero-hph-PtefA-pepA recombinant vector.

[0050] Example 3: Transformation and Screening of Aspergillus niger 1. Protoplast preparation: 1.1 Fresh Aspergillus niger ATCC 1015 spores were inoculated into 100 mL of liquid PDA+UU medium and cultured at 28℃ and 150 rpm for 12 h.

[0051] 1.2. Transfer 10 mL of bacterial cells into a 50 mL centrifuge tube and centrifuge at 8000 rpm for 10 min, then discard the supernatant.

[0052] 1.3. Add the enzymatic hydrolysate (the preparation process of the enzymatic hydrolysate is: dissolve 20 mg Yatalase and 30 mg Lysing Enzymes in 10 mL Osmotic medium) to a centrifuge tube, resuspend it, and then transfer it to a 50 mL Erlenmeyer flask for enzymatic hydrolysis for 3 h. It is necessary to blow the tube once during the middle to ensure that the strain in the bottom plate is fully enzymatically hydrolyzed. The protoplasts are then examined under an optical microscope.

[0053] 1.4 Transfer the enzymatically digested product to a 50 mL centrifuge tube. Slowly add 10 mL of Trapping Buffer close to the tube wall. Centrifuge at 5000 rpm for 5 min at 4 °C. Transfer the protoplasts from both liquid surfaces to another 50 mL centrifuge tube. Dilute the protoplast suspension with an equal volume of STC buffer. Centrifuge at 6000 rpm for 5 min at 4 °C. Discard the supernatant. Resuspend the protoplasts in 1 mL of STC buffer to obtain the protoplast solution. Place on ice.

[0054] 2. Transformation: 2.1 Take a sterile 1.5 mL centrifuge tube and mix the recombinant vectors p-zero-pyrG-PgpdA-amyA and p-zero-pyrG-PgpdA-pepA obtained in Example 2 with 70 μL of protoplast solution. Incubate on ice for 50 min to obtain a mixture.

[0055] 2.2 Take a sterile 15 mL centrifuge tube, add the mixture to the 15 mL centrifuge tube, then add 1.5 mL of PEG4000 (polyethylene glycol 4000) solution, mix gently, let stand at room temperature for 20 min, then add 10 mL of transformation medium, mix well, and plate for culture to obtain transformants. Aspergillus niger 1015 transformed with the recombinant vector p-zero-pyrG-PgpdA-amyA was obtained. amyA Aspergillus niger 1015 transformed into the recombinant vector p-zero-pyrG-PgpdA-pepA pepA .

[0056] 2.3. Subsequently, prepare Aspergillus niger 1015 according to the above steps. amyA The protoplasts were transformed into Aspergillus niger 1015 via the recombinant vector p-zero-hph-PtefA-pepA. amyA, pepA .

[0057] 3. Validation and preservation of positive transformants: 3.1. Using sterile toothpicks, the transformed spores were further spotted onto a solid plate of PDA and incubated at 28°C for 2 days.

[0058] 3.2 Then, pick the transformed spores and hyphae into a 1.5 mL EP tube, add 500 μL Lysis Buffer, and heat in a 60℃ metal bath for 2 h.

[0059] 3.3 Add 150 μL of KAC buffer, vortex until homogeneous, and then centrifuge at 12000 rpm for 5 min.

[0060] 3.4 After centrifugation, transfer the supernatant to a new 1.5 mL EP tube, add an equal amount of isopropanol, and then centrifuge at 12000 rpm for 5 min.

[0061] 3.5 After discarding the supernatant, add 300 μL of 70% ethanol to wash the DNA, and then centrifuge at 12000 rpm for 5 min.

[0062] 3.6 After the ethanol has dried, add 50 μL of DEPC (diethyl pyrocarbonate) to treat the water and dissolve the DNA.

[0063] 3.7. PCR verification of the single clone genome. If correct, pick a small number of spores from the hyphae and inoculate the target strain using the dense streak method. After culturing for 2 days, collect the spores with sterile water and then preserve the fresh spores in 25% glycerol.

[0064] Example 4: Performance Verification of the Strains RT-PCR (Reverse Transcription Polymerase Chain Reaction) and qPCR (Quantitative Real-Time Polymerase Chain Reaction) Detection: During the semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection, total RNA was extracted using the Spin Column Fungal Total RNA Purification Kit (Sangon Biotech, B518659). Subsequently, following the kit's recommended steps, the RNA was reverse transcribed into cDNA using HiScriptII Q RT SuperMix for qPCR (+gDNA wiper). Each PCR reaction was initiated with 50 ng of cDNA template. To amplify the target gene fragment, the following thermal cycling conditions were used: DNA denaturation at 95℃, followed by annealing and extension at 60℃, for a total of 24 cycles. Using the *Aspergillus niger* tubA gene as an internal control, the relative expression levels of the amyA and pepA genes were detected to confirm the overexpression effect.

[0065] Fresh Aspergillus niger 1015 prepared in Example 2, Aspergillus niger 1015amyA Aspergillus niger 1015 pepA and Aspergillus niger 1015 amyA，pepA Conidia (concentration 1×10) 8 (Spores / mL) were inoculated into a 250 mL Erlenmeyer flask containing 50 mL of seed culture medium, and then the mixture was incubated at 28°C with shaking at 200 rpm for 20 hours. Afterwards, 50 mL of the mixture was transferred to another Erlenmeyer flask containing 8 g of starch and incubated for another 16 hours to promote spore formation. Finally, total RNA was extracted from the cultured mycelium using a Spin Column Fungal Total RNA Purification Kit (Sangon Biotech, B518659), and reverse transcribed into cDNA using a HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Novizan, R223-01). Quantitative real-time PCR analysis (QPCR) was performed using QuantStudio. TM 3. Real-time PCR was performed using the ChamQ Blue Universal SYBR qPCR Master Mix kit (Novizan, product number Q311-02) on a real-time PCR system (manufactured by Applied Biosystems, Inc., USA). The expression levels of the target gene were detected using the comparative threshold cycling method (ΔCT method), and normalized based on the expression level of the tubA gene; relative transcript levels (… Figure 1 The 15.2 times indicated on the bar chart is achieved through 2 ΔΔCT It is calculated using a formula.

[0066] Growth phenotype observation: After each strain was activated on a PDA plate, it was inoculated onto glucose-MM medium and starch-MM medium with sterile toothpicks and cultured at 28℃ for 3 days. The size and morphology of the colonies were observed.

[0067] Starch degradation capacity determination: *Aspergillus niger* strains preserved in 25% glycerol were inoculated onto PDA solid medium and cultured at 28℃ for 3 days. An appropriate amount of sterile water was added, and the spores were gently washed with a spreader to obtain a spore suspension. An appropriate amount of the spore suspension was diluted to a suitable factor, and the spores were counted under an electron microscope using a hemocytometer to calculate the concentration of the spore suspension. The known concentration (1×10⁻⁶) was then used to determine the spore concentration. 8A spore suspension (spores / mL) was inoculated into seed culture medium and cultured at 28°C and 200 rpm for 20 h. Then, 5 mL of the seed culture was inoculated into fermentation medium and fermented with shaking at 28°C and 200 rpm. Each shake-flask fermentation was repeated three times. Every 12 h or 24 h, 1 mL of sample was taken to measure starch consumption (100 μL of Gram's iodine solution (1 g iodine and 2 g potassium iodide in 300 mL distilled water) was added, shaken to mix, and the starch consumption was judged based on the color difference; the deeper the blue of the mixture, the higher the remaining starch content in the system; as the reaction time increased, if the color of the mixture gradually lightened until it became colorless, it indicated that the starch had been completely consumed. Acid production detection: After fermentation, 2 mL of fermentation broth was added to a 50 mL centrifuge tube, along with an equal volume of 2 M hydrochloric acid, and allowed to stand for 10 min for acidification. 2 mL of the acidified solution was then added to a 2 mL centrifuge tube and centrifuged at 12,000 rpm for 10 min. The supernatant was diluted 20-50 times, filtered through a 0.22 μm aqueous filter membrane, and the concentrations of succinic acid and citric acid were determined by HPLC.

[0068] 5. Results and Analysis qPCR results as follows Figure 1 The results showed that the single-gene Aspergillus niger 1015 amyA Aspergillus niger 1015 pepA The expression levels of the corresponding amyA and pepA genes in the *Aspergillus niger* strain 1015 were significantly higher than those in the original strain. amyA,pepA Both genes are expressed efficiently.

[0069] Growth phenotype experiments such as Figure 2 As shown, the starting strain ATCC1015 grows slowly on starch-MM medium, and the single-gene Aspergillus niger 1015 amyA Aspergillus niger 1015 pepA Small colonies, Aspergillus niger C colonies (double-gene recombinant Aspergillus niger 1015) amyA,pepA The diameter is significantly larger than that of single-gene strains, and the growth status is good.

[0070] Starch hydrolysis analysis revealed that strain Aspergillus niger 1015 amyA and Aspergillus niger 1015 pepA They already possessed a certain starch hydrolysis capacity. However, after 84 hours of cultivation, neither AmyA nor PepA completely consumed the starch, but this is sufficient to prove that AmyA and PepA did indeed play a role in the starch hydrolysis process of Aspergillus niger. Based on this, this study constructed a dual-gene overexpression strain of Aspergillus niger 1015. amyA,pepA The result is as follows Figure 3 The results showed that the strain had a significantly enhanced starch hydrolysis capacity and a significantly shortened starch depletion time, requiring only 66 hours to completely consume the starch.

[0071] Acid production test results as follows Figure 4 The results showed that the dual-gene Aspergillus niger produced succinic acid up to 15.6 g / L and citric acid up to 12.3 g / L in a fermentation medium with starch as the sole carbon source. Both of these yields were higher than those of the starting strain ATCC 1015 in a fermentation medium with glucose as the carbon source (9.2 g / L and 5.6 g / L, respectively).

[0072] Shake-flask fermentation experiment: In actual shake-flask fermentation, it was observed that the starting strain ATCC 1015 exhibited severe mycelial aggregation and was difficult to disperse in a fermentation medium containing 10% by weight starch (semi-solid after sterilization); while the double-gene Aspergillus niger 1015... amyA,pepA Under the same conditions, the mycelia were well dispersed and the culture medium was in a uniform suspension state.

[0073] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A strain of Aspergillus niger ( Aspergillus niger ), characterized in that, The Aspergillus niger was obtained by simultaneously overexpressing α-amylase and aspartic protease in the starting strain.

2. The Aspergillus niger according to claim 1 ( Aspergillus niger ), characterized in that, The originating strain has the accession number ATCC 1015.

3. The Aspergillus niger (A) according to claim 1 spergillus niger ), characterized in that, The nucleotide sequence of the gene encoding the α-amylase is shown in SEQ ID NO. 1; And / or, the nucleotide sequence of the gene encoding the aspartic protease is shown in SEQ ID NO.

2.

4. The Aspergillus niger (A) according to claim 3 spergillus niger ), characterized in that, The Aspergillus niger ( Aspergillus niger The device contains promoter I for overexpressing the gene encoding α-amylase and promoter II for overexpressing the gene encoding aspartic protease; the genes encoding the overexpressing α-amylase and aspartic protease are provided in the form of a recombinant vector.

5. The Aspergillus niger according to claim 4 ( Aspergillus niger ), characterized in that, The promoter I is the Pgpd promoter; And / or, the promoter II is the Ptef promoter.

6. A type of Aspergillus niger ( Aspergillus niger The construction method of ) is characterized by, The method includes overexpressing the encoding genes for α-amylase and aspartic protease in the starting strain.

7. The method according to claim 6, characterized in that, The Aspergillus niger ( Aspergillus niger The accession number for this is ATCC 1015; And / or, the nucleotide sequence of the gene encoding the α-amylase is shown in SEQ ID NO. 1; And / or, the nucleotide sequence of the gene encoding the aspartic protease is shown in SEQ ID NO. 2; And / or, the Aspergillus niger ( Aspergillus niger The device contains promoter I for overexpressing the gene encoding α-amylase and promoter II for overexpressing the gene encoding aspartic protease; the genes encoding α-amylase and aspartic protease for overexpression are provided in the form of a recombinant vector. And / or, the promoter I is a Pgpd promoter; And / or, the promoter II is the Ptef promoter.

8. The Aspergillus niger as described in any one of claims 1-5 ( Aspergillus niger ) and / or the method described in claim 6 or 7 to construct Aspergillus niger ( Aspergillus niger Application in the degradation of starch to produce citric acid and / or succinic acid.

9. A method for producing citric acid and / or succinic acid, characterized in that, The method includes: applying any one of the Aspergillus niger molds described in claims 1-5 (… Aspergillus niger ) and / or the method described in claim 6 or 7 to construct Aspergillus niger ( Aspergillus niger The fermentation culture is carried out; the carbon source for the fermentation culture is starch, and the amount of starch used is 70-110 g relative to 1 L of the fermentation culture medium used for the fermentation culture.

10. The method according to claim 9, characterized in that, The fermentation conditions include: a time of 55-70 h, a temperature of 20-35℃, and a rotation speed of 180-220 rpm.

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