Application of mutant topoisomerase II coding gene in enhancing acid resistance of fungi

Abstract

The invention discloses an application of a mutant topoisomerase II coding gene in enhancing the acid resistance of fungi, and belongs to the field of molecular biology. According to the invention, point mutation is carried out on the topoisomerase II coding gene of the model fungus Aspergillus nidulans, and the topoisomerase II structure is partially damaged, so that the acid resistance of the Aspergillus nidulans is enhanced, and the addition amount of a neutralizing agent in the production of L-malic acid is further reduced. The method is a brand new method capable of enhancing the acid resistance of the fungi, and can effectively solve the problems that the cost is increased and waste is generated to pollute the environment due to the fact that the existing fungi cannot tolerate a low-pH environment and a neutralizer needs to be continuously added in the organic acid production process.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and particularly relates to the application of a mutant topoisomerase II encoding gene in enhancing the acid resistance of fungi. Background Art

[0002] L-malic acid, a dicarboxylic acid found ubiquitously in all organisms, is chemically known as 2-hydroxysuccinic acid. Due to its strong sour flavor and pleasant mouthfeel, it has gradually replaced citric acid as the most widely used organic acidulant and flavor enhancer in the food, beverage, and confectionery industries. Furthermore, malic acid and its derivatives also play important roles in pharmaceuticals, chemicals, biology, and materials science. Currently, industrial production of L-malic acid primarily utilizes filamentous fungi such as Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei, and yeasts such as Saccharomyces cerevisiae and Pichia pastoris. However, during fermentation, these microorganisms require the continuous addition of neutralizing agents such as calcium carbonate to the fermenter to maintain a near-neutral fermentation environment; otherwise, L-malic acid production plummets. The large amount of neutralizing agent added results in the generation of large amounts of waste salts during downstream separation and purification of the L-malic acid. These waste salts have very low utility and are often disposed of in landfills. Disposal of these waste salts accounts for more than half of the total production cost. Therefore, reducing or eliminating the addition of neutralizing agents has become a major challenge in the industrial production of L-malic acid.

[0003] At present, relevant literature reports have been released. Researchers have obtained acid-resistant strains in yeast through natural separation or adaptive laboratory evolution. These acid-resistant strains can grow normally in a low pH environment, and it has been found that these acid-resistant strains can reduce neutralizers during the production of L-malic acid. However, there are currently few research reports on the ability of acid-resistant strains to produce L-malic acid and reduce neutralizers, and they are limited to yeast. The disadvantage is that their acid resistance has not yet met the ideal industrial requirements and the acid resistance mechanism is unclear. Filamentous fungi have always been the main industrial strains for fermenting and growing malic acid due to their advantages in industrial-scale malic acid production. Among them, Aspergillus is the most commonly known strain for producing L-malic acid. However, due to its weak acid resistance, calcium carbonate needs to be added. These problems have always been the pain points of industrial fermentation production. Therefore, it is of great significance to invent a method to enhance the adaptation of Aspergillus fungi to acidic environments, thereby reducing the use of neutralizers. Summary of the Invention

[0004] Purpose of the invention: In response to the problems existing in the prior art, the present invention provides an application of topoisomerase II in enhancing the acid resistance of strains. The present invention enhances the acid resistance of fungi by mutating the topoisomerase II encoding gene. This is a newly discovered gene mutation method that can enhance the acid resistance of fungi. It can effectively solve the problem that existing fungi cannot tolerate low pH environments and need to continuously add neutralizers during the organic acid production process, thereby causing environmental pollution.

[0005] The present invention also provides the genetic engineering bacteria for enhancing the acid resistance of fungi by destroying topoisomerase II and its application.

[0006] Technical solution: In order to achieve the above purpose, the present invention uses the topoisomerase II in enhancing the acid resistance of fungi, the encoding gene of the topoisomerase II is as shown in SEQ ID NO.1, or the encoding gene of a homologous protein with more than 80% homology to the topoisomerase II.

[0007] The fungus is a fungus having the topoisomerase II or a homologous protein with more than 80% homology to the topoisomerase II, including Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae or Trichoderma reesei filamentous fungi.

[0008] Wherein, the acid resistance of the strain is enhanced by mutating the topoisomerase II domain or an important site of its homologous protein in the fungus.

[0009] Among them, the acid resistance of the strain was enhanced by mutating the topoisomerase II encoding gene in the fungus.

[0010] Preferably, the fungus has a topoisomerase II encoding gene, and mutating the topoisomerase II encoding gene can enhance the fungus's resistance to acidic environmental stress.

[0011] Furthermore, a point mutation is performed on the topoisomerase II encoding gene of Aspergillus nidulans to partially damage the structure of topoisomerase II, thereby enhancing the adaptability of Aspergillus nidulans to an acidic environment.

[0012] Preferably, the Aspergillus nidulans is Aspergillus nidulans FGSC A4

[0013] Among them, by performing point mutation on the topoisomerase II encoding gene of Aspergillus nidulans, the structure of topoisomerase II is partially damaged, thereby enhancing the acid resistance of Aspergillus nidulans and reducing the amount of additives used in the production of L-malic acid.

[0014] Among them, the application of enhancing the acid resistance of Aspergillus nidulans to reduce the amount of neutralizer added during L-malic acid production is achieved by mutating the 421st base "C" to "T"; and / or the 568th base "C" to "T" in the topoisomerase II encoding gene SEQ ID NO.1.

[0015] Wherein, the neutralizing agent is calcium carbonate.

[0016] The invention discloses a mutant topoisomerase II for enhancing the acid resistance of fungi. The coding gene of the mutant topoisomerase II is characterized by a mutation of the 421st base C to T and / or a mutation of the 568th base C to T in the gene shown in SEQ ID NO.1.

[0017] The acid-resistant genetically engineered bacteria of the present invention are obtained by mutating the topoisomerase II encoding gene shown in SEQ ID NO.1 or the encoding gene of a homologous protein with more than 80% homology to the topoisomerase II, and the mutation of the topoisomerase II encoding gene is performed by mutating the 421st base C in the gene shown in SEQ ID NO.1 to T and / or mutating the 568th base C to T.

[0018] The mutant topoisomerase II for enhancing the acid resistance of fungi or the acid-resistant genetically engineered bacteria of the present invention are used in enhancing the acid resistance of fungi or producing L-malic acid.

[0019] Among them, the mutant topoisomerase II or acid-resistant genetically engineered bacteria that enhances the acid resistance of fungi are used in the production of L-malic acid while reducing or not adding a neutralizing agent.

[0020] The topoisomerase II encoding gene described in the present invention is the gene numbered XM_657211.1 on the NCBI website. Furthermore, the topoisomerase II-related gene sequence is XM_657211.1 or a gene encoding a homologous protein in other filamentous fungi corresponding to the protein it encodes. The nucleotide sequence of the gene XM_657211.1 (ANIA_04699) is shown in SEQ ID NO. 1.

[0021] Among them, the mutant sequence of the point mutation is derived from an acid-resistant strain obtained through adaptive laboratory evolution screening in an acidic environment; the adaptive laboratory evolution screening process is divided into two stages, the first stage is acidic solid environment screening and the second stage is acidic liquid environment screening.

[0022] Furthermore, the acid-resistant genetically engineered Aspergillus nidulans strain of the present invention is obtained by mutating the topoisomerase II encoding gene using Aspergillus nidulans as a base strain.

[0023] The invention relates to the use of the acid-resistant genetically engineered Aspergillus nidulans bacteria in producing L-malic acid.

[0024] The acid-resistant genetically engineered Aspergillus nidulans is used to produce L-malic acid while reducing or eliminating the need for adding a neutralizing agent.

[0025] Furthermore, the acid-resistant genetically engineered Aspergillus nidulans can improve its acid resistance in both acidic solid culture medium and liquid culture medium, and both the acidic solid culture medium and liquid culture medium are basic culture media for maintaining the growth of the strain.

[0026] The present invention is the first to mutate the topoisomerase II encoding gene in Aspergillus nidulans to enhance the acid resistance of Aspergillus nidulans so as to reduce the amount of neutralizers such as calcium carbonate added in the production of L-malic acid.

[0027] The method of the present invention for enhancing the acid resistance of fungi by mutating the topoisomerase II encoding gene comprises the following steps:

[0028] Using whole-genome resequencing technology, five acid-resistant Aspergillus nidulans mutant strains and two parental strains obtained through adaptive laboratory evolution screening were analyzed. After verification through Sanger first-generation sequencing, a gene (ANIA_04699) mutated in both acid-resistant strains was screened out. This gene contains a conserved domain of the topoisomerase II-related protein PATⅠ.

[0029] Using point mutagenesis technology, point mutations were made to the gene in Aspergillus nidulans, partially damaging the conserved domain of the topoisomerase II-related protein PATⅠ.

[0030] The acid resistance of the mutant strain was compared with that of the parent strain by spotting the bacteria on acidic solid culture medium and culturing on acidic liquid culture medium and statistically analyzing the colony diameter, biomass and other data of the mutant strain and the parent strain.

[0031] The present invention utilizes point mutations in this gene to generate mutant strains that enhance their adaptability to acidic environments. The present invention utilizes homologous recombination gene editing technology to construct point mutation strains, which are then verified using Sanger sequencing. The present invention also verifies the acid resistance of the point mutation strains by spotting the strains on acidic solid culture media and by shaking the strains on liquid culture media.

[0032] Specifically, in order to screen acid-resistant Aspergillus nidulans strains, an adaptive laboratory evolution experiment was designed. The specific steps included:

[0033] 1. An adaptive laboratory evolution screening process was performed using Aspergillus nidulans engineered strains PL03-3 and PL03-4 that had undergone five gene editings (up-regulating key genes in the rTCA cycle: pyruvate carboxylase gene pyc (SEQ ID NO.3), malate dehydrogenase gene mdhC (SEQ ID NO.4), and C4-dicarboxylate transporter gene dctA (SEQ ID NO.5) that promotes the export of L-malate to the extracellular space; up-regulating key genes in the glycolysis pathway: glucose transporter gene mstE (SEQ ID NO.6), 6-phosphofructokinase gene pfkA (SEQ ID NO.7), the rate-limiting step in the third step of glycolysis, and knocking out oxaloacetate acetylhydrolase gene oahA (SEQ ID NO.8), citrate transporter gene cexA (SEQ ID NO.9), and pyruvate decarboxylase gene pdcA (SEQ ID NO.10) (see Example 1 for details of the specific gene editing process).

[0034] 2. In the first stage, strains PL03-3 and PL03-4 were preliminarily induced and enriched on a solid culture medium with gradually decreasing pH adjusted by L-malic acid (500 g / L), and acid-resistant strains were preliminarily screened.

[0035] 3. In the second stage, the acid-resistant strains preliminarily screened out above were cultured in shake flasks in a liquid culture medium with gradually decreasing pH adjusted by L-malic acid (500 g / L). After screening, 5 acid-resistant strains were finally retained (named: PL03-3-10, PL03-3-16, PL03-4-11, PL03-4-16 and PL03-4-17).

[0036] The invention discloses an application of enhancing the acid resistance of Aspergillus nidulans by mutating topoisomerase II to reduce the neutralizing agent calcium carbonate in the production of L-malic acid.

[0037] The present invention constructs a point mutant strain by mutating topoisomerase II, enhancing the strain's acid resistance. Shake flask fermentation of the acid-resistant strain to produce L-malic acid reduces the amount of calcium carbonate used by half while maintaining acid production. This is the first time that screening for an acid-resistant strain in Aspergillus nidulans has reduced the use of neutralizing agents in organic acid production.

[0038] This study, using whole-genome resequencing analysis of an acid-resistant Aspergillus nidulans strain that underwent adaptive laboratory evolution in an acidic environment and verified using Sanger sequencing technology, identified key sites in a gene containing the conserved domain of the topoisomerase II-associated protein PAT I, which is associated with acid resistance. By performing point mutations on this gene, the conserved domain of the topoisomerase II-associated protein PAT I was altered, enhancing the acid resistance of Aspergillus nidulans. This approach provides an effective approach for enhancing acid resistance in fungal strains and reducing the need for calcium carbonate when producing organic acids using fungi.

[0039] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0040] The present invention provides a method for enhancing the acid resistance of fungi by mutating the topoisomerase II encoding gene, and finds that the mutated topoisomerase II encoding gene can enhance the acid resistance of fungi such as Aspergillus nidulans and reduce the amount of neutralizer added during the production of L-malic acid in Aspergillus nidulans.

[0041] The method described in the present invention can be applied to other fungi containing important sites of topoisomerase II or its homologous proteins, providing a new method for reducing the use of neutralizers in the process of producing organic acids by other industrial strains, and is of great significance to the process of producing organic acids by microbial fermentation in the field of synthetic biology.

[0042] The present invention also constructs two highly acid-resistant Aspergillus nidulans genetically engineered bacteria (named PL04 ​​and PL05). Compared with the control strain PL03-4, which has an L-malic acid yield of 43.98 g / L when 40 g / L of calcium carbonate, a neutralizer, is added during fermentation for 192 hours, the L-malic acid yields of the highly acid-resistant Aspergillus nidulans genetically engineered bacteria are 40.71 g / L and 39.35 g / L, respectively, after fermentation for 192 hours. Moreover, the amount of calcium carbonate added as a neutralizer can be reduced to 20 g / L, a reduction of 50%. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Schematic diagram of the plasmids pCZ02 and pPL01 / PL02 described in the present invention.

[0044] Figure 2 Figures show the results of overexpressing the pfkA gene and knocking out the pdcA gene in the ZQ08 strain described in the present invention. (A) Schematic diagram of the CRISPR-Cas9 gene editing-mediated knockout of the pfkA and pdcA genes. (B) Diagnostic PCR validation results of the transformed strains using the primer pair PdcA-gc-yz-F / R. Lanes 1 / 2 / 3 show the results of the PL01 strain, and lane 4 shows the PCR results of the control strain ZQ07. (C) Relative expression levels of the pfkA gene mRNA regulated by the Ptef promoter.

[0045] Figure 3 Schematic diagram of the Cre-loxP-mediated screening marker recovery system described in the present invention. (A) Brief overview of the Cre-loxP system. The Pxylp-Cre-pryoA band is flanked by two loxP sites in the same direction. After induction with xylose, the fragment between the two loxP sites is lost, leaving only one loxP site. (B) PCR verification of the induced strain was performed using primers set on both sides of the two loxP sites. The bands in lanes 1 and 2 are 5166 bp, and there are no bands in lanes 3 and 4, indicating that pyroA has been lost. (C) Phenotypic diagram of PL01 and PL02 on MMPR and MMR culture media.

[0046] Figure 4 Analysis of the results of overexpressing the glucose transporter gene mstE described in the present invention. (A) Schematic diagram of the overexpressed Ptef-mstE-loxP-Pxylp-Cre-pyroA-loxP fragment. (B) Agarose gel electrophoresis of PCR diagnostics of four randomly selected transformants. (C) Schematic diagram of the metabolic pathways involved in the synthesis of L-malic acid in Aspergillus nidulans. Red and blue fonts represent knockout and overexpressed genes, respectively. PdcA: pyruvate decarboxylase; PfkA: 6-phosphofructokinase; MstE: glucose transporter. (D) Phenotypic images of ZQ01, PL01, and PL03 strains grown on MMPR and MMR media.

[0047] Figure 5 Schematic diagram of the adaptive laboratory evolution screening process described in the present invention. (A) Schematic diagram of the process for induction and enrichment screening of acid-resistant strains in solid culture medium and liquid shake flasks with continuously decreasing pH. (B) Phenotype of the parent strains (PL03-3, PL03-4) and five acid-resistant strains (PL03-3-10, PL03-3-16, PL03-4-11, PL03-4-16, and PL03-4-17) on acidic solid MMPR culture medium with pH of 6.5 and 2.65, respectively. (C) Biomass statistics of the parent strains (PL03-3, PL03-4) and five acid-resistant strains (PL03-3-10, PL03-3-16, PL03-4-11, PL03-4-16, and PL03-4-17) in acidic liquid MMPR culture medium with a pH of 2.80 (ns represents no significant difference).

[0048] Figure 6This is a diagram showing the sequencing results of the mutation site of the mutant gene ANIA_04699 of the acid-resistant strains PL03-4-11 (the 421st base "C" mutated to "T") and PL03-4-17 (the 568th base "C" mutated to "T") described in the present invention.

[0049] Figure 7 Domain and amino acid sequence conservation analysis of the XP_662303.1 protein described in the present invention. (A) Domain analysis of the XP_662303.1 protein in Aspergillus nidulans FGSC A4. (B) Amino acid sequence conservation analysis of mutation sites in the XP_662303.1 protein sequence across Aspergillus species.

[0050] Figure 8 Graphs illustrating the point mutation construction process and phenotypic analysis described herein, as well as L-malic acid production analysis results for the point mutant strains. (A) Schematic diagram of the principle of point mutation construction mediated by homologous recombination technology. (B) Biomass statistics of the control strains PL03-4, PL03-4-11, and PL03-4-17 and the point mutant strains PL04 ​​and PL05 in a liquid shake flask at pH 2.90. (C) Phenotypic plots of the control strains PL03-4, PL03-4-11, and PL03-4-17 and the point mutant strains PL04 ​​and PL05 on solid culture media at pH 6.5 and 2.65, respectively. (D) L-malic acid production and pH change results for the control strain PL03-4 after 192 hours of fermentation with the addition of 40 g / L calcium carbonate. (E) L-malic acid production and pH change results for the point mutant strain PL04 ​​after 192 hours of fermentation with the addition of 20 g / L calcium carbonate. (F) L-malic acid production and pH change results of the point mutant strain PL05 after fermentation for 192 h with the addition of 20 g / L calcium carbonate. DETAILED DESCRIPTION

[0051] The present invention will be further described below with reference to the accompanying drawings and examples.

[0052] Unless otherwise specified, the materials and reagents used in the examples of the present invention can be obtained from commercial sources. Experimental methods without specific conditions in the examples are generally performed under conventional conditions or the conditions recommended by the manufacturer.

[0053] Aspergillus nidulans FGSC A4 was provided by Nanjing Normal University. Localization and function of calmodulin in live-cells of Aspergillus nidulans, Fungal Genetics and Biology 47 (2010) 268–278.

[0054] Aspergillus nidulans TN02A7, provided by Nanjing Normal University, Localization and function of calmodulin in live-cells of Aspergillus nidulans, Fungal Genetics and Biology 47 (2010) 268–278.

[0055] The culture medium formula used in the embodiment is as follows:

[0056] YAG liquid medium (1 L): Glucose 20 g, yeast extract 5 g, trace elements 1 mL. Solid medium: Add 20 g agar.

[0057] MMPR basic medium (1 L): Glucose 10 g, 20× Nitrate Salts 50 mL, 1000× Trace elements 1 mL, Vitamin B2 (riboflavin) 6.6 μM, Vitamin B6 (pyridoxine) 2.5 μM, NaOH to adjust pH to 6.5, agar 20 g.

[0058] MMR basic medium (1 L): glucose (Glucose) 10 g, 20× Nitrate Salts 50 mL, 1000× trace elements (Trace elements) 1 mL, vitamin B2 (riboflavin) 6.6 μM, NaOH to adjust the pH to 6.5, agar (Agar) 20 g.

[0059] MM minimal medium (1 L): Glucose 10 g, 20× Nitrate Salts 50 mL, 1000× Trace elements 1 mL, NaOH to adjust pH to 6.5, agar 20 g.

[0060] SMMR screening medium (1 L): Glucose 10 g, 20× Nitrate Salts 50 mL, Sorbitol 218.6 g, 1000× Trace elements 1 mL, Vitamin B2 (riboflavin) 6.6 μM, NaOH to adjust pH to 6.5, agar 7.5 g.

[0061] SMM screening medium (1 L): Glucose 10 g, 20× Nitrate Salts 50 mL, Sorbitol 218.6 g, 1000× Trace elements 1 mL, NaOH to adjust pH to 6.5, agar 7.5 g.

[0062] XMMPR xylose induction medium (1 L): xylose 10 g, 20× Nitrate Salts 50 mL, 1000× trace elements 1 mL, vitamin B2 (riboflavin) 6.6 μM, vitamin B6 (pyridoxine) 2.5 μM, NaOH to adjust pH to 6.5, agar 20 g.

[0063] 2×MMPR acidic liquid medium (1 L): Glucose 20 g, 20× Nitrate Salts 100 mL, 1000× Trace elements 2 mL, Vitamin B2 (riboflavin) 13.2 μM, Vitamin B6 (pyridoxine) 5.0 μM, L-malic acid (500 g / L) to adjust the pH.

[0064] 2× Water Agar (1 L): Agar 40 g.

[0065] LB medium (1 L): 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride (NaCl), and 20 g of agar for solid culture medium.

[0066] LA medium (1 L): LB medium supplemented with 1 / 1000 volume fraction of ampicillin (Amp).

[0067] Construction of high expression plasmids pPL01 and pPL02:

[0068] According to the patent: A CRISPR-Cas9-mediated simultaneous dual gene editing system and its application in Aspergillus (Application No. 202310756480.3), the pCZ02 expression plasmid was constructed. This plasmid contains the strong Tef promoter, the trpC terminator, two loxP sites, the Pxylp promoter, the Cre recombinase gene fragment, and the pyroA nutritional selection marker gene fragment (length 6051bp).

[0069] To overexpress the target genes pfkA and mstE, plasmids pPL01 and pPL02 were constructed as follows: the genome of Aspergillus nidulans TN02A7 was extracted in large quantities and used as a template. The pfkA and mstE gene fragments were amplified using primer pairs 0-pfkA-F / Tef-pfkA-R and 0-mstE-F / Tef-mstE-R, respectively, and cloned into the Ptef promoter gene fragment of pCZ02, resulting in plasmids pPL01 and pPL02. The Ptef promoter gene fragment was oriented in the same direction as the pfkA and mstE gene fragments, and in the opposite direction to the other original sequences. The plasmid schematic is shown in Figure 2. Figure 1 .

[0070] Transformation of protoplasts:

[0071] (1) Collect the spores of the strain grown on YAG solid medium with 0.2% Tween 80, rinse twice with sterile water, count with a hemocytometer, and take 2×10 8 spores were added to 100 mL of liquid MMPR medium.

[0072] (2) Cultivate at 28°C and 220 rpm, blow evenly, aspirate the mycelium and place it on a glass slide, and observe the spore germination state under a microscope. When most of the spores have germinated and the tooth tube is within the length of 1-2 spores (about 8-9 hours), it is the optimal growth state and can be used for subsequent experiments.

[0073] (3) Prepare the same protoplast enzymatic solution as above, filter and sterilize it, and use it immediately. Filter and sterilize the prepared enzymatic solution (the enzyme is fully dissolved, the solution is transparent and has no particle feeling) with a sterile double-layer filter membrane with a pore size of 0.22 μm, and place it in a sterile conical flask (50 mL). At the same time, break up the mycelium cultured in the shake flask in step (2) (you can oscillate and vortex to ensure that the mycelium is fully spread out), let it settle, draw 10 mL into a 50 mL sterile centrifuge tube, and centrifuge it at 8000 rpm for 10 minutes to precipitate the mycelium. Discard the culture medium completely, and transfer the precipitated mycelium to 10 mL of protoplast enzymatic solution, shake and mix thoroughly to ensure the best enzymatic effect, and enzymatically hydrolyze for 4 hours at 28 ° C and 80 rpm.

[0074] (4) After about 2 hours, gently blow the enzymatic solution to break up the mycelium again. After 4 hours, gently pipette a small amount of enzymatic solution onto a glass slide and observe the state of the protoplasts under a microscope. If obvious vacuoles are present throughout the entire viewing angle, the subsequent experimental steps can be carried out.

[0075] (5) Slowly transfer all the enzymatic hydrolysate products with suitable growth status to a 50mL sterile centrifuge tube (operate on ice), add 10mL of solution 2 (Trapping Buffer) drop by drop along the wall of the tube, and centrifuge at 4℃, 5000rpm for 10min. After centrifugation, the centrifuge tube will show obvious stratification. Gently aspirate 10mL of milky white protoplasts in the middle and a small amount of hyphae gathered below, place them in a new 50mL sterile centrifuge tube, continue to slowly add an equal volume of solution 3 (STC Buffer) along the wall of the tube on ice, and centrifuge at 4℃, 6000rpm for 10min. The obtained protoplasts are attached to the wall of the centrifuge tube, discard most of the supernatant, observe the amount of protoplasts under a microscope, add an appropriate volume of STC Buffer according to the number of protoplasts in the field of view, and resuspend to obtain protoplasts. The prepared protoplast suspension is stored on ice for later use.

[0076] (6) Pipette an appropriate volume of protoplasts (100-150 μL) into a sterile centrifuge tube, add 10 μg of the target DNA fragment, gently pipette to mix, and place on ice for 50 minutes.

[0077] (7) Take out a new 15mL sterile centrifuge tube and add 1.5mL solution 4 (PEG buffer), transfer the above protoplast and DNA mixture to the centrifuge tube, gently flick to mix and let it stand at room temperature for 25 minutes. Add 10mL of the prepared transformation medium to a clean sterile culture dish, and solidify to obtain the transformation lower layer. Take another 10mL to a 50mL sterile centrifuge tube, wait for the culture medium to cool, add it to the mixed tube of protoplasts and DNA, turn it upside down to mix, quickly pour it on the solidified lower layer culture medium and shake it lightly to spread it out, and obtain the transformation upper layer. Place it on a clean bench until it is completely solidified, incubate it in a 37℃ incubator for 2-3 days, and obtain transformants for subsequent analysis.

[0078] Solutions and culture media used for transformation:

[0079] Solution 1 (OM): 1.2 M MgSO4, 10 mM NaPB buffer (90.9 g Na2HPO4, 163.4 g NaH2PO4 per liter of 2M NaPB buffer), adjust pH to 5.8 with 1 M Na2HPO4, and store at 4°C.

[0080] Solution 2 (Trapping Buffer): 0.6 M sorbitol, 0.1 M Tris-HCl pH 7.0, sterilize at 115°C for 30 min, and store at 4°C.

[0081] Solution 3 (STC Buffer): 1.2 M sorbitol, 10 mM CaCl2, 10 mM Tris-HCl pH 7.5, sterilize at 115°C for 30 min, and store at 4°C.

[0082] Solution 4 (PEG): 60% PEG 4000, 50 mM CaCl2, 50 mM Tris-HCl pH 7.5, sterilized at 115°C for 30 min and stored at room temperature.

[0083] Protoplastization enzymatic hydrolysis solution: 10 mL OM, 30 mg Lysing Enzymes from Trichoderma, 20 mg Yatalase, filter sterilize, prepare and use immediately.

[0084] Miniprep of Aspergillus nidulans genome:

[0085] (1) A small amount of spores / mycelium was inoculated into a 1.5 mL centrifuge tube containing 500 μL of liquid YAG medium and cultured at 37°C and 220 rpm for 24 h;

[0086] (2) After the spores have grown into mycelial balls, centrifuge at 12,000 rpm for 5 minutes. Discard the supernatant, add 500 μL of lysis buffer, and lyse the spores at 60°C for 30-60 minutes.

[0087] (3) Take out a 1.5 mL centrifuge tube, add 150 μL of KAC solution, vortex, and centrifuge at 12000 rpm for 5 min to precipitate impurities.

[0088] (4) 400 μL of the supernatant after centrifugation was aspirated and transferred to a new 1.5 mL centrifuge tube. An equal volume of isopropanol reagent was then added and centrifuged at 12,000 rpm for 5 min.

[0089] (5) Pour out all the liquid, add 300 μL of 75% ethanol solution, and centrifuge at 12000 rpm for 2-5 minutes.

[0090] (6) Pour out all the liquid and dry the excess liquid in the centrifuge tube at 60°C. Add 30-50 μL of sterile water, measure the concentration, and store at -20°C until use.

[0091] The steps for gene transcription level analysis are as follows:

[0092] 1. RNA extraction:

[0093] RNA from Aspergillus nidulans was extracted using the Sangon column-based total RNA extraction kit UNIQ-10 (B511361). All reagents included in the kit were used. The entire extraction process was performed in a windless and dust-free environment. The specific steps are as follows:

[0094] (1) Inoculation and collection of bacteria. Collect fresh spores and inoculate 1×10 8 Incubate the culture medium with 100 spores in liquid MMPR medium at 37°C for 48 h. Filter the medium through a double-layer filter cloth, rinse with sterile water, absorb excess water with clean filter paper, and immediately freeze in liquid nitrogen.

[0095] (2) Grind the bacterial powder. Add 450 mL of RLT Solution to a 1.5 mL RNA-free EP tube and grind the mycelium with liquid nitrogen to obtain fine bacterial powder. Before the liquid nitrogen evaporates completely, transfer the powder to an EP tube and vortex thoroughly to mix. Centrifuge at 12,000 rpm at 4°C for 5 min.

[0096] (3) Pipette 400 μL of the supernatant after centrifugation into a new 1.5 mL RNA-free EP tube and add 200 μL of anhydrous ethanol. Invert the tube, mix gently, and place on ice.

[0097] (4) Combine the adsorption column and collection tube, transfer all the mixed liquid to the center of the adsorption column, let it stand on ice for 2 minutes, and centrifuge it at 8000 rpm at 4°C for 1 minute.

[0098] (5) Discard the centrifugation solution, add 500 μL of RW Solution along the tube wall, let it stand on ice for 1 min, and centrifuge at 10,000 rpm at 4°C for 1 min.

[0099] (6) Discard the centrifuged solution and add 500 μL of RPE Solution along the tube wall. After the sample is placed on ice for 2 minutes, centrifuge it at 10,000 rpm at 4°C for 1 minute, discard the filtrate, and repeat this step.

[0100] (7) Dry the adsorption column at 4°C, 10,000 rpm, and vacuum for 2 min.

[0101] (8) Place the adsorption column in a new 1.5 mL RNA-free EP tube, add 30-50 μL of DEPC water, and incubate on ice for 5 min. Centrifuge at 12,000 rpm for 2 min at 4°C. Measure the concentration and store the resulting RNA solution at -80°C.

[0102] 2. RT-qPCR experiment:

[0103] (1) The RNA content extracted above needs to be within 500 ng. According to m / c=v, calculate the required volume of RNA and add it on ice according to Table 1 below. Perform PCR reaction according to Table 2:

[0104] Table 1 Reaction system

[0105]

[0106]

[0107] Table 2 Synthesis-related PCR procedures

[0108]

[0109] The lid temperature was set to 90°C and the obtained cDNA was stored at -20°C until use.

[0110] (2) RT-qPCR reaction system was added on ice according to Table 3 below:

[0111] Table 3 RT-qPCR reaction system

[0112]

[0113] (3) Subsequent experimental steps were performed according to the instructions of ABI one-step fast thermocycler (Applied Biosystems).

[0114] (4) The endogenous tubA gene of Aspergillus nidulans was selected as the internal reference gene, and the relative expression level was calculated using the ΔΔCT algorithm.

[0115] The primers used in the examples of the present invention are shown in Table 4 below. The amplification PCR reaction system, fusion PCR reaction program, and PCR reaction program during the construction of the point mutation strain are shown in Tables 5-7, respectively:

[0116] Table 4 Primer list

[0117]

[0118]

[0119] Table 5 Amplification PCR reaction system

[0120]

[0121]

[0122] Table 6 Fusion PCR reaction program

[0123]

[0124] Table 7 PCR reaction program

[0125]

[0126] Example 1

[0127] Five gene editing operations were performed on the wild-type Aspergillus nidulans strain TN02A7 to construct the engineered strain PL03.

[0128] The starting strain used in the examples of the present invention was Aspergillus nidulans strain TN02A7 (WT), provided by Nanjing Normal University. Localization and function of calmodulin in live-cells of Aspergillus nidulans, Fungal Genetics and Biology 47 (2010) 268–278. The first three gene editing steps are detailed in the patent: A CRISPR-Cas9-mediated simultaneous dual-gene editing system and its application in Aspergillus (Application No. 202310756480.3). After three gene editing steps and Cre-loxP system-mediated selection marker recovery, strain ZQ08 was subjected to a second round of gene editing.

[0129] Based on the recipient strain ZQ08, the pdcA gene was knocked out and the pfkA gene was overexpressed as a repair template to construct the PL01 strain. Figure 2 As shown in A.

[0130] The specific construction process is as follows:

[0131] 1. In vitro synthesis of pdcA gene targeting sgRNA:

[0132] (1) Synthesize double-stranded DNA template of sgRNA. The reaction system is as shown in Table 8:

[0133] Table 8 DNA template synthesis system

[0134]

[0135]

[0136] (2) PCR amplification of DNA template. The PCR program is as shown in Table 9:

[0137] Table 9 DNA template synthesis related PCR program

[0138]

[0139] (3) Agarose gel electrophoresis was used to verify and recover the target DNA (120 bp in size) using the Gel Extraction Kit (a two-in-one DNA recovery kit from Novozymes). The DNA concentration was then determined to serve as the subsequent dsDNA template DNA.

[0140] (4) Transcription and synthesis of sgRNA: Transcription and synthesis of sgRNA was performed according to the instructions of T7 MEGAscript kit (Life Technologies). The reaction system is as shown in Table 10:

[0141] Table 10 sgRNA in vitro transformation system

[0142]

[0143] (5) RNA purification. After the reaction is completed, add 115 μL DEPC water, 15 μL Ammonium Acetate Stop Solution, 115 μL phenol-chloroform-isopropanol mixture and 300 μL chloroform in sequence. The mixed solution is centrifuged at 4°C and 13,000g for 15 minutes. Pipette 150 μL of the clear upper aqueous phase into a new RNA-free EP tube, add 150 μL of isopropanol solution, and centrifuge at 4°C and 13,000g for 10 minutes. Discard the upper liquid, retain the precipitate, add 1000 μL of 75% ethanol, and centrifuge at 4°C and 13,000g for 6 minutes. Discard the supernatant, retain the precipitate, and place it in an open position in a clean bench for 10 minutes to dry. Add DEPC water according to the amount of precipitate, measure the concentration, and store the synthesized sgRNA in a -80°C ultra-low temperature refrigerator for later use.

[0144] 2. Synthesize the repair template:

[0145] The repair DNA template (Ptef-pfkA-loxP-Pxylp-Cre-pyroA-loxP) was amplified using plasmid pPL01, and the nick created by the sgRNA-Cas9 complex was inserted. The repair template includes the constitutive promoter Ptef, the nutritional selection marker pyroA, the Cre-loxP system, the target gene pfkA, and approximately 30 bp of microhomology arms from the pdcA gene. To prevent the constitutive promoter from interfering with other genes, the promoter was designed in the opposite direction to the pfkA gene during amplification. Using the designed upstream primer (PdcA-0-tpc-F) and downstream primer (PdcA-0-tpc-R), the entire repair template, including the approximately 30 bp of microhomology arms, was amplified from plasmid pPL01. Band size was verified by DNA gel electrophoresis. Correct bands were recovered using a gel recovery kit and stored at -20°C until further use.

[0146] 3. Preparation and transformation steps of Aspergillus nidulans protoplasts:

[0147] (1) Use 0.2% Tween 80 to collect ZQ08 strain spores grown on YAG solid medium, rinse twice with sterile water, count using a hemocytometer, and take 2×10 8 spores were added to 100 mL of liquid MMPR medium.

[0148] (2) Cultivate at 28°C and 220 rpm, blow evenly, aspirate the mycelium and place it on a glass slide, and observe the spore germination status under a microscope. When most of the spores have germinated and the germ tube is within the length of 1 to 2 spores (about 8-9 hours), it is in the optimal growth state and can be used for subsequent experiments.

[0149] (3) Prepare the protoplastization enzymatic solution, filter and sterilize it, and use it immediately. Filter and sterilize the prepared enzymatic solution (the enzyme is fully dissolved, the solution is transparent and has no particle feeling) with a sterile double-layer filter membrane with a pore size of 0.22 μm, and place it in a sterile conical flask (50 mL). At the same time, break up the mycelium cultured in the shake flask in step (2) (you can oscillate and vortex to ensure that the mycelium is fully spread out), let it settle, draw 10 mL into a 50 mL sterile centrifuge tube, and centrifuge it at 8000 rpm for 10 minutes to precipitate the mycelium. Discard the culture medium completely, and transfer the precipitated mycelium to 10 mL of the protoplastization enzymatic solution. Thoroughly shake and mix to ensure the best enzymatic hydrolysis effect, and enzymolyze at 28 ° C and 80 rpm for 4 hours.

[0150] (4) After about 2 hours, gently blow the enzymatic solution to break up the mycelium again. After 4 hours, gently pipette a small amount of enzymatic solution onto a glass slide and observe the state of the protoplasts under a microscope. If obvious vacuoles are present throughout the entire viewing angle, the subsequent experimental steps can be carried out.

[0151] (5) Slowly transfer all the enzymatic hydrolysate products with suitable growth status to a 50mL sterile centrifuge tube (operate on ice), add 10mL of solution 2 (Trapping Buffer) drop by drop along the tube wall, and centrifuge at 4℃, 5000rpm for 10min. After centrifugation, the centrifuge tube will show obvious stratification. Gently aspirate 10mL of milky white protoplasts in the middle and a small amount of hyphae gathered below, place them in a new 50mL sterile centrifuge tube, continue to slowly add an equal volume of solution 3 (STC Buffer) along the tube wall on ice, centrifuge at 4℃, 6000rpm for 15min. After centrifugation, the protoplasts will stick to the tube wall. Discard most of the supernatant and observe the amount of protoplasts under a microscope. Add an appropriate volume (100-200μL) of STC Buffer according to the number of protoplasts in the field of view and resuspend to obtain protoplasts. The prepared protoplast suspension is stored on ice for later use.

[0152] (6) Pipette an appropriate volume of protoplasts (150 μL) into a sterile centrifuge tube, add 4 μg of the purified sgRNA and 5 μg of the amplified DNA repair template, gently pipette to mix, and place on ice for 50 min.

[0153] (7) Take out a new 15mL sterile centrifuge tube and add 1.5mL solution 4 (PEG buffer), transfer the above protoplast and DNA mixture to the centrifuge tube, gently flick to mix and let it stand at room temperature for 25 minutes. Add 10mL of the prepared SMMR transformation medium to a clean sterile culture dish and solidify to obtain the transformation lower layer. Take another 10mL to 50mL sterile centrifuge tube, wait for the culture medium to cool slightly, add it to the above protoplast and DNA mixing tube, turn it upside down to mix, quickly pour it on the solidified lower layer culture medium and shake it lightly to spread it out, and obtain the transformation upper layer. Place it on the clean bench until it is completely solidified, incubate it in a 37℃ incubator for 2-3 days, and obtain transformants for subsequent analysis.

[0154] 4. Select transformants, perform diagnostic PCR verification, and perform transcriptional analysis:

[0155] After 2-3 days of incubation, three transformants were randomly picked from the transformation plate for diagnostic PCR. The primers (PdcA-gc-yz-F and PdcA-gc-yz-R) were designed at both ends of the gene pdcA, and their size was 2574 bp. If the repair template was successfully integrated into the gap generated by the sgRNA-Cas9 protein cutting, a band of 9704 bp in length would be obtained by PCR verification ( Figure 2 B). The band sizes shown by agarose gel electrophoresis were consistent with the expected values, indicating that the repair template (Ptef-pfkA-loxP-Pxylp-Cre-pyroA-loxP) had been integrated into the pdcA locus in all three selected transformants. At the same time, analysis of the high-expression gene pfkA at the mRNA level showed that ( Figure 2 C), the expression level of the pfkA gene in the successfully constructed strain PL01 was 2.26 times that of the control strain.

[0156] Using the Cre-loxP-mediated selection marker recovery system described in the patent "A CRISPR-Cas9-mediated simultaneous dual gene editing system and its application in Aspergillus," the pyroA nutritional selection marker was recovered from the newly constructed strain PL01 to construct strain PL02. In this system, the Cre recombinase can specifically recognize the 34bp loxP site and catalyze the recombination between two loxP sites in the same direction, thereby deleting the DNA fragment between the loxP sites, including the nutritional selection marker. The working principle diagram is shown below. Figure 3(A) The gene encoding Cre recombinase is regulated by a xylose-inducible promoter (Pxylp), and two equidirectional loxP sites are located on either side, forming part of the repair template. The newly constructed PL01 strain was induced and incubated on a basic medium containing xylose (three-zone, 37°C, cultured for 2 days). Xylose can induce the discarding of pyroA. Single colonies that cannot grow on MMR medium but can grow on MMPR medium are preliminarily considered to have discarded the fragment containing the selection marker pyroA between the two loxP sites. The strain is called PL02. After culture, a small amount of genome was extracted from a single colony, and primers (Cre-loxp-F and Cre-loxp-R) were set on both sides of the two loxP sites for PCR diagnosis and verification. The length of the Pxylp-Cre-pryoA fragment is 5166bp, as shown in the figure. Figure 3 As shown in (B), the PCR results showed that the Pxylp-Cre-pryoA fragment was present in the PL01 strain, while this fragment was lost in the PL02 strain, which included the nutritional selection marker pyroA. The results were further verified by spotting the bacteria on MMPR and MMR solid plates. Figure 3 As shown in (C), the induced PL02 strain can grow normally on the medium supplemented with pyridoxine, but cannot grow normally on the medium without pyridoxine, indicating that pyroA in the PL02 strain has indeed been lost and can be used as a recipient strain for subsequent gene editing.

[0157] The strain PL02 recovered by nutritional screening marker was used as the recipient strain to highly express the glucose transporter mstE gene ( Figure 4 C) First, the constructed plasmid pPL02 with the high-profile mstE gene was used as a template, and the primer pair O-mstE-F and M13-R were used to amplify the fragment Ptef-mstE-loxP-Pxylp-Cre-pyroA-loxP, which was then introduced into the recipient bacteria (the detailed transformation process is the same as above). Figure 4 A shows a high-profile repair template fragment. Four transformants were randomly selected from the transformation plate and amplified using the 0-mstE-F and 0-tef-R primer pairs. The results are shown in Figure 4 As shown in B, it shows that the Ptef-mstE fragment has been integrated into the recipient bacteria. The four transformants of the constructed engineered strain were named PL03-1, PL03-2, PL03-3, and PL03-4. The results of spotting on MMPR and MMR solid culture media showed that overexpression of the mstE gene had no significant effect on the phenotype of the strain ( Figure 4 D).

[0158] Example 2

[0159] Adaptive laboratory evolution was performed on Aspergillus nidulans engineered strains PL03-3 and PL03-4, which had undergone five gene editings (to upregulate key genes in the rTCA cycle: pyruvate carboxylase gene pyc, malate dehydrogenase gene mdhC, and C4-dicarboxylate transporter gene dctA that promotes L-malate export to the extracellular space; to upregulate key genes in the glycolysis pathway: glucose transporter gene mstE, 6-phosphofructokinase gene pfkA, the rate-limiting step in the third step of glycolysis, and to knock out oxaloacetate acetylhydrolase gene oahA, citrate transporter gene cexA, and pyruvate decarboxylase gene pdcA). Figure 5 A).

[0160] 1. Prepare acidic MMPR solid culture medium by mixing the pH-adjusted gradient acidic MMPR liquid culture medium (pH = 4.0, 3.8, 3.6, 3.4, 3.2, 3.0, and 2.8) with 2× water agar in a 1:1 ratio and then pouring the mixture onto plates to obtain acidic solid MMPR culture medium with different gradients.

[0161] 2. Engineered strains PL03-3 and PL03-4 were plated on a gradient of acidic solid MMPR medium. Based on the strains' growth in the medium, a pH of 3.2 was determined as the initial pH for the first phase of solid-adaptive laboratory evolution screening. The pH of the acidic solid MMPR medium was then gradually lowered, and colonies that grew on the acidic solid MMPR medium were spotted on media with even lower pH values. Ultimately, several acid-tolerant strains of PL03-3 and PL03-4 were identified on solid MMPR medium at pH 2.35.

[0162] 3. Because colonies in acidic liquid culture media are more completely encapsulated by the acidic environment, the acid-resistant strains obtained above were further screened by shaking in a second phase of acidic liquid MMPR culture media. The acid-resistant strains PL03-3 and PL03-4 obtained above were shaken in a gradient of acidic liquid MMPR culture media. Based on the growth of the strains in the culture media, pH = 3.0 was determined as the initial pH value for the second phase of adaptive laboratory evolution. By gradually lowering the pH of the culture media and continuously subculturing, after 10 subculturing cycles, five strains that tolerated liquid MMPR culture media at pH = 2.8 were retained and named: PL03-3-10, PL03-3-16, PL03-4-11, PL03-4-16, and PL03-4-17.

[0163] 4. If Figure 5As shown in (B) and (C), the acid-tolerant strains were spotted on acidic solid MMPR medium. The results showed that the acid-tolerant strains screened were more acid-tolerant than the parent strains PL03-3 and PL03-4. Biomass statistics also showed no significant difference in biomass between the acid-tolerant strains and the parent strains, demonstrating that the acid-tolerant strains can indeed grow normally in acidic liquid environments.

[0164] Example 3

[0165] The acid-resistant strains screened were subjected to whole genome resequencing and the parent strains were analyzed.

[0166] 1. Inoculate 5 acid-resistant strains (PL03-3-10, PL03-3-16, PL03-4-11, PL03-4-16, PL03-4-17) and 2 parent strains (PL03-3, PL03-4) with 1×10 8 The spores were transferred to 100 mL of MMPR liquid medium and cultured at 37°C and 220 rpm for 1 day.

[0167] 2. After 1 day, collect the mycelium with gauze, wrap it with tin foil, quickly freeze it with liquid nitrogen, and then freeze it in a -80℃ refrigerator.

[0168] 3. Entrust Shanghai LuMing Biotechnology Co., Ltd. (referred to as "Ouyi Biomass Spectrometry") to conduct whole genome resequencing analysis on the above 7 Aspergillus nidulans samples.

[0169] 4. According to the results of whole genome sequencing, the genomes of the seven strains were extracted, and primers ANIA_04699-F and ANIA_04699-R were designed for amplification. After Sanger sequencing, a gene ANIA_04699 was screened out that was mutated in both the PL03-4-11 and PL03-4-17 strains. The gene sequence is shown in SEQ ID NO.1, but the mutation sites in the gene of the two strains are different. The 421st "C" base of the ANIA_04699 gene of the PL03-4-11 strain mutated to a "T" base, and the corresponding amino acid mutated from CAG (glutamine) to UAG (stop codon). The mutation site is located in the PAT1 domain. The 568th "C" base of the ANIA_04699 gene of the PL03-4-17 strain mutated to a "T" base, and the corresponding amino acid mutated from CGG (arginine) to UGG (tryptophan) ( Figure 6 ).

[0170] Example 4

[0171] Analysis of amino acid sequence conservation at XP_662303.1 protein sequence mutation sites in filamentous fungi

[0172] 1. The amino acid sequence of the protein corresponding to the gene represented by SEQ ID NO. 1 of Aspergillus nidulans FGSC A4 was analyzed by blastp analysis on NCBI. Sequences highly homologous to some of the amino acid sequences of Aspergillus nidulans were found (including Aspergillus oryzae RIB40, Aspergillus niger ATCC1015, and Trichoderma reesei QM6a), and an amino acid sequence alignment was drawn.

[0173] 2. If Figure 7 As shown in (A), there is a PAT I (topoisomerase II) domain in the XP_662303.1 protein sequence, which belongs to the topoisomerase II superfamily and is related to DNA damage repair. Figure 7 As shown in (B), the two mutated amino acid sites in the amino acid sequence corresponding to the ANIA_04699 gene are conserved in filamentous fungi such as Aspergillus oryzae and Aspergillus niger, indicating that sequences with similar functions may also exist in other species, and that the protein can be stably expressed during translation and expression without random mutations.

[0174] Example 5

[0175] The parent strain PL03-4 was subjected to a point mutation in gene ANIA_04699 to construct a point mutation strain

[0176] 1. Use homologous recombination gene editing technology to perform point mutations ( Figure 8A). First, the genomes of the acid-resistant strains PL03-4-11 (mutation of "C" at position 421 to "T") and PL03-4-17 (mutation of "C" at position 568 to "T") were extracted, and a 1102-bp fragment upstream of the ANIA_04699 gene (left homology arm) was amplified using primer pairs ANIA_04699-P1 and ANIA_04699-P3. Primers ANIA_04699-P4 and ANIA_04699-P6 were used to amplify a 1724-bp DNA fragment (right homology arm) encompassing the upstream portion of the ANIA_04699 gene and a portion of the gene fragment, including the two mutation sites of the ANIA_04699 gene. The left and right homology arms each contained a sequence that complemented the start and end positions of the selection marker ribo gene (SEQ ID NO. 2), enabling ligation to the selection marker gene sequence. The genome of Aspergillus nidulans R21 (provided by Nanjing Normal University, Localization and function of calmodulin in live-cells of Aspergillus nidulans, Fungal Genetics and Biology 47 (2010) 268–278) was used as a template to amplify the ribo nutritional selection marker fragment using the primer pair Ribo-F and Ribo-R. The left homology arm, the ribo selection marker gene, and the right homology arm were mixed at a molar ratio of 1:2:1 using the primer pair ANIA_04699-P2 and ANIA_04699-P5. The three fragments were fused, and the fragment sizes were determined by diagnostic PCR amplification and agarose gel electrophoresis analysis. The specific sequences of the three fusion fragments for the two mutation methods are shown in SEQ ID NO. 11 (base 421 "C" mutated to "T") and SEQ ID NO. 12 (base 568 "C" mutated to "T"), respectively.

[0177] 2. The three fused fragments were mixed with protoplasts of the parent strain PL03-4 and incubated on ice for 50 min. 10 μg of the fragments were added to every 100 μL of protoplasts, followed by 1.25 mL of PEG and allowed to stand at room temperature for 25 min. The strains were screened using SMM medium, a screening medium without pyridoxine (riboflavin). The colonies that grew were cultured in rich YAG medium and genomic DNA was extracted. PCR was performed using primers ANIA_04699-F and ANIA_04699-P5. The amplified fragments were subjected to Sanger sequencing to verify whether they were point mutation strains. The correct point mutation strains were named PL04 ​​(the 421st base "C" of the ANIA_04699 gene was mutated to "T") and PL05 (the 568th base "C" of the ANIA_04699 gene was mutated to "T").

[0178] 3. Phenotypic tests were conducted on the constructed point mutant strains PL04 ​​and PL05. Gradient dilution spot tests were performed on acidic solid MMPR medium at pH = 2.65. The results showed that the colony forming units of the point mutant strains PL04 ​​and PL05 were 10 times higher than those of the control strain PL03-4 ( Figure 8 C). At the same time, the biomass results in the liquid shake flask at pH = 2.90 also showed that the control strain PL03-4 could hardly grow in the acidic environment, while the biomass of the point mutant strain had no significant difference compared with the acid-resistant strains PL04-11 and PL04-17 ( Figure 8 B) This indicates that the acid resistance of the point mutation strain is significantly stronger than that of the control strain.

[0179] Example 6

[0180] The L-malic acid production fermentation test of the point mutant strain can reduce the amount of calcium carbonate added as a neutralizing agent

[0181] Currently, in the process of producing L-malic acid by microbial fermentation, neutralizing agents such as calcium carbonate need to be continuously added to the culture medium to maintain the fermentation pH near neutral. The addition of large amounts of neutralizing agents will cause a large amount of waste salt to be generated during the downstream separation and purification of L-malic acid, which not only pollutes the environment but also greatly increases the production cost of L-malic acid produced by microbial fermentation.

[0182] The specific acid production conditions are as follows:

[0183] 1. 1×10 8Fresh spores were inoculated into 50 mL seed culture medium containing 30 g / L glucose, 0.6 g / L KH2PO4, 2 g / L urea, 0.5 g / L MgSO4·7H2O, 0.11 g / L ZnSO4·7H2O, 0.088 g / L FeSO4·7H2O and 0.1% Yeastextract and cultured at 37°C and 220 rpm for 24 h. Then, the mycelial pellets were transferred to fermentation medium containing 80 g / L glucose, 0.6 g / L KH2PO4, 0.2 g / L urea, 0.5 g / LMgSO4·7H2O, 0.11 g / L ZnSO4·7H2O, 0.088 g / L FeSO4·7H2O, 0.1% Yeast extract and 1 g / L CaCO3 to produce L-malic acid and cultured at 37°C and 220 rpm for 8 days. During the period, samples were taken every 2 days. 1 g / L CaCO3 was added to the fermentation medium of the parent strain PL03-4 each time a sample was taken. The point mutant strains PL04 ​​and PL05 only added 1 g / L CaCO3 to adjust the pH when sampling on the 2nd day, and no additional CaCO3 was added thereafter.

[0184] 2. Acid production by Aspergillus nidulans was analyzed using high-performance liquid chromatography (HPLC). An equal volume of 6 M HCl was added to 1 mL of fermentation broth to neutralize excess calcium carbonate and acidify the broth. The broth was centrifuged at 12,000 rpm for 10 minutes. The supernatant was aspirated and filtered through a 0.22 μm filter. The resulting sample was stored at 4°C for subsequent experiments.

[0185] 3. For organic acid detection conditions, refer to Table 11 below:

[0186] Table 11 HPLC detection conditions

[0187]

[0188]

[0189] like Figure 8 As shown in Figures (D-F), the L-malic acid production of the point mutant strains PL04 ​​and PL05 constructed above was compared with that of the parent strain PL03-4 using shake flask fermentation, as well as the addition of the neutralizing agent calcium carbonate. The results showed that the control strain PL03-4, with the normal addition of 40 g / L of the neutralizing agent calcium carbonate, produced 43.98 g / L of L-malic acid after 192 hours of fermentation. Compared to the control strain, the newly constructed point mutant strains PL04 ​​and PL05 achieved L-malic acid production of 40.71 g / L and 39.35 g / L, respectively, and were able to reduce the addition of the neutralizing agent calcium carbonate to 20 g / L, a 50% reduction.

Claims

1. Application of topoisomerase II in enhancing the acid resistance of fungi, wherein the gene encoding the topoisomerase II is as shown in SEQ ID NO. 1, or the gene encoding a homologous protein with more than 80% homology to the topoisomerase II.

2. The use according to claim 1, characterized in that The fungus is a fungus having the topoisomerase II or a homologous protein with more than 80% homology to the topoisomerase II, and the fungus includes Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae or Trichoderma reesei filamentous fungi.

3. The use according to claim 1, characterized in that In the fungus, the acid resistance of the strain is preferably enhanced by mutating the important sites of the topoisomerase II domain or its homologous protein.

4. The use according to claim 1, characterized in that The acid resistance of the strain is enhanced by mutating the topoisomerase II encoding gene in the fungus.

5. The use according to claim 4, characterized in that By performing point mutation on the topoisomerase II encoding gene of Aspergillus nidulans, the structure of topoisomerase II is partially damaged, thereby enhancing the acid resistance of Aspergillus nidulans and reducing the amount of neutralizer added during L-malic acid production.

6. The use according to claim 5, characterized in that The invention discloses an application of enhancing the acid resistance of Aspergillus nidulans and reducing the amount of calcium carbonate added as a neutralizing agent during L-malic acid production by mutating the 421st base "C" to "T" and / or the 568th base "C" to "T" in the topoisomerase II encoding gene SEQ ID NO.

1.

7. A mutant topoisomerase II that enhances the acid resistance of fungi, characterized in that: The coding gene of the mutant topoisomerase II is mutated from the 421st base C to T and / or the 568th base C to T in the gene shown in SEQ ID NO.

1.

8. An acid-resistant genetically engineered bacterium, characterized in that: The genetically engineered strain is obtained by using a fungus having a topoisomerase II encoding gene as shown in SEQ ID NO.1 or a gene encoding a homologous protein with more than 80% homology to the topoisomerase II as a base strain, and mutating the topoisomerase II encoding gene. The mutation of the topoisomerase II encoding gene is to mutate the 421st base C in the gene shown in SEQ ID NO.1 to T, and / or mutate the 568th base C to T.

9. Use of the mutant topoisomerase II for enhancing the acid resistance of fungi according to claim 7 or the acid-resistant genetically engineered bacterium according to claim 8 in enhancing the acid resistance of fungi or in producing L-malic acid.

10. The use according to claim 9, characterized in that The mutant topoisomerase II or acid-resistant genetically engineered bacteria capable of enhancing the acid resistance of fungi is used in producing L-malic acid while reducing or eliminating the addition of a neutralizing agent.

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