CN111088173A - Aspergillus niger genetically engineered bacterium and construction method and application thereof - Google Patents

Abstract

The invention discloses an Aspergillus niger genetically engineered bacterium, which is Aspergillus niger for knocking off a glucan synthesis regulation gene VosA. The invention also discloses a construction method of the genetic engineering bacteria, and further discloses application of the genetic engineering bacteria in production of citric acid through immobilized fermentation. According to the invention, aspergillus niger genetically engineered bacteria of a gene knockout glycan synthesis regulation gene VosA are constructed, so that the hydrophobicity of aspergillus niger is reduced and the yield of a biological membrane is reduced in the process of producing citric acid by immobilized fermentation, the agglomeration phenomenon of a carrier is reduced, the yield of citric acid and the sugar conversion efficiency are improved, and the method is suitable for industrial production.

Description

Aspergillus niger genetically engineered bacterium and construction method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to an Aspergillus niger genetically engineered bacterium for knocking out a glucan synthesis regulation gene VosA, and a construction method and application thereof.

Background

Citric acid is one of the most demanded organic acids worldwide, and is widely used in industries such as food, medicine, daily chemicals and the like. Wherein, 75% of citric acid is used in food industry, mainly used in sour agent, antioxidant, pH regulator in food additive, meanwhile, since citric acid has mild and refreshing sour taste, it is widely used in the manufacture of food such as beverage, cake, wine, dairy products, etc. Another 15% is used in the chemical and textile industries and can be used as buffers, complexing agents, sequestering agents, metal builders, mordants, etc. Also 10% of citric acid is used as anticoagulant, antacid, taste corrigent, cosmetic and feed additive, etc., and is widely used in the pharmaceutical industry and animal husbandry. In recent years, due to the general rise of food prices in China, the cost of raw materials for producing citric acid is greatly increased. Therefore, how to improve the citric acid fermentation method and further reduce the cost becomes the most concerned problem in the whole citric acid industry in China.

The citric acid produced by fermenting aspergillus niger is one of the main ways for producing citric acid by current industrial fermentation, and the fermentation efficiency of aspergillus niger can be greatly improved by a cell immobilization method. At present, there are four main methods for cell immobilization: entrapment, cross-linking, covalent bonding, and adsorption. For filamentous fungi, the adsorption method is simple to operate, the chemical structure is not required to be changed in the fixing process, and the method is theoretically suitable for all filamentous fungi and is more beneficial to industrial popularization and application. The principle of the adsorption method mainly depends on the interaction force between the groups on the surface of the carrier and the surface of the cells, so that the cells are promoted to be adsorbed on the surface of the carrier for growth. And when the cells die, the cells fall off from the carrier, and simultaneously, the active cells are adsorbed to form dynamic balance, so that the whole microenvironment forms an efficient conversion system, and the overall fermentation efficiency is obviously improved compared with the free fermentation. In the process of research on the immobilized fermentation mechanism, microorganisms commonly form a biological membrane on a carrier, and the formed biological membrane is different, so that the performance of immobilized fermentation is greatly different.

Disclosure of Invention

The purpose of the invention is as follows: in order to solve the problems of low yield and long fermentation period in the existing industrial production of citric acid by Aspergillus niger fermentation, the invention provides an Aspergillus niger genetically engineered bacterium of glucan synthesis regulation gene VosA defect type, and provides a construction method of the Aspergillus niger genetically engineered bacterium and application of the Aspergillus niger genetically engineered bacterium in preparation of citric acid by fermentation.

The technical scheme is as follows: in the first aspect of the invention, the aspergillus niger genetically engineered bacterium is provided, and a glucan synthesis regulation gene VosA in the aspergillus niger genetically engineered bacterium is inactivated.

The Aspergillus niger genetically engineered bacterium is a genetically engineered bacterium which replaces a partial sequence of a gene VosA by a hyg resistance gene (hygromycin resistance gene) through a double exchange method, wherein the gene VosA can identify and combine a VRE region (CTTATCCGGCC) in a promoter of an Fks (glucan synthase) family gene so as to regulate and control the expression of the Fks family gene, and the gene negatively regulates and controls the Fks family gene, can inhibit the transcription of Fks after being combined with the promoter, so that the synthesis of β -1,3 glucan is inhibited, the cell wall thickness and the extracellular integrity are further influenced, the hydrophobic layer is deleted, the hydrophobicity of Aspergillus niger is reduced, the adsorption performance and the formation of a biological membrane are greatly weakened, and the immobilized fermentation effect and the yield of citric acid are influenced.

Preferably, the original Aspergillus Niger is Aspergillus Niger ATCC 12846.

Preferably, the inactivated nucleotide sequence of the glucan synthesis control gene VosA is shown in SEQ ID NO. 2, and the inactivated nucleotide sequence of the glucan synthesis control gene VosA is shown in SEQ ID NO. 1.

The second aspect of the invention provides a construction method of the aspergillus niger genetically engineered bacterium, which comprises the following steps:

(1) extracting the original genomic DNA of the Aspergillus niger;

(2) amplifying to obtain an upstream homology arm and a downstream homology arm of the gene VosA by using the genome DNA obtained in the step (1) as a template; amplifying to obtain a hygromycin resistance gene by taking a plasmid PAN7-1 as a template; taking an upstream homology arm and a downstream homology arm of the gene VosA and a hygromycin resistance gene as templates, and performing overlap extension PCR amplification to obtain a gene knockout fragment;

(3) and (3) introducing the gene knockout fragment obtained in the step (2) into an Aspergillus niger protoplast for homologous recombination to obtain the Aspergillus niger genetically engineered bacterium with the inactivated glucan synthesis regulatory gene VosA.

Preferably, in step (1) the original Aspergillus Niger is Aspergillus Niger ATCC 12846;

preferably, the nucleotide sequences shown in SEQ ID NO 3 and SEQ ID NO 4 are used as primers when the upstream homologous arm of the gene VosA is amplified in the step (2); the nucleotide sequences shown in SEQ ID NO 5 and SEQ ID NO 6 are used as primers when the downstream homologous arm of the gene VosA is amplified; when the hygromycin resistance gene is amplified, the nucleotide sequences shown in SEQ ID NO. 7 and SEQ ID NO. 8 are used as primers; when the PCR amplification gene knockout segment is subjected to overlap extension, the nucleotide sequences shown in SEQ ID NO. 9 and SEQ ID NO. 10 are used as primers.

Preferably, the nucleotide sequence of the upstream homology arm of the gene VosA is shown as SEQ ID NO. 11, the nucleotide sequence of the downstream homology arm of the gene VosA is shown as SEQ ID NO. 12, the nucleotide sequence of the hygromycin resistance gene is shown as SEQ ID NO. 13, and the nucleotide sequence of the gene knockout fragment is shown as SEQ ID NO. 14.

The third aspect of the invention provides application of the Aspergillus niger genetically engineered bacterium in fermentation preparation of citric acid.

Preferably, the aspergillus niger genetically engineered bacteria are used as fermentation strains to prepare the citric acid through immobilized fermentation.

Preferably, the immobilized fermentation takes porous fiber material as an immobilized medium.

More preferably, the porous fibrous material is carbon black fiber.

The method for producing citric acid by immobilized fermentation of aspergillus niger genetically engineered bacteria with inactivated glucan synthesis regulatory gene VosA specifically comprises the following steps:

(1) preparing a carrier: soaking the porous fiber material in absolute ethyl alcohol for 1-1.5 h, then washing with water until no obvious ethyl alcohol taste exists, and drying to constant weight to obtain a modified porous fiber material; cutting into pieces of the same size and volume of 0.5cm³A cubic block carrier of (1).

(2) Fermentation: and scraping the activated aspergillus niger genetically engineered bacterium flat plate by using a sporulation liquid to prepare a spore liquid. Inoculating the mixture into a fermentation medium of sterilized porous fiber materials in an inoculation amount of 0.4% (v/v), and fermenting to obtain the citric acid.

Preferably, the preparation method of the fermentation medium for immobilized fermentation comprises the following steps of weighing corn flour, adding water to prepare corn flour liquid with the concentration of 200g/L-300g/L, adding 0.5mL-1mL of liquefying enzyme into each 1L of fermentation liquid, preserving the heat at 60 ℃ to 75 ℃ for 35min-45min, heating the corn flour liquid to 85 ℃ to 105 ℃, adding 0.5mL-1mL of liquefying enzyme into each 1L of fermentation liquid, carrying out enzymolysis for 45min-60min until the iodine solution does not turn blue, filtering the corn flour liquid to obtain corn clear liquid, adding unfiltered corn flour liquid into the corn clear liquid, mixing the unfiltered corn flour liquid and the corn clear liquid in a volume ratio of 2-10:100, and uniformly mixing to obtain the immobilized fermentation medium, wherein the liquefying enzyme contains α -amylase, and the enzyme activity of α -amylase is 70000U/mL (the enzyme activity of 1mL of 1min for hydrolyzing 1mg of soluble starch is 1U/mL of enzyme activity unit under the condition that the 1mL of enzyme liquid is 5.5.5.5 ℃ and 85 ℃.

Preferably, the fermentation conditions of the immobilized fermentation are as follows: the fermentation temperature is 30-37 ℃, the fermentation time is 72-115 h, and the fermentation rotating speed is 200-330 rpm.

Preferably, the porous fiber material is a modified porous fiber material, and the modification steps are as follows: soaking the porous fiber material in absolute ethyl alcohol for 1-1.5 h, washing with water until no obvious ethyl alcohol taste exists (3-5 times), and drying to constant weight to obtain the modified porous fiber material. The amount of the porous fiber material and the absolute ethyl alcohol is not particularly required during soaking, and the soaking purpose is achieved.

Has the advantages that: the invention constructs an Aspergillus niger genetically engineered bacterium with a glucan synthesis regulatory gene VosA deletion by a gene knockout means. The genetic engineering bacteria reduce the amount of biomembrane in the process of producing citric acid by aspergillus niger through immobilized fermentation, effectively relieve the phenomenon that the pore diameter of a carrier is blocked, and increase the oxygen and mass transfer effects of the carrier and the outside, thereby exerting the advantages of immobilized fermentation, improving the yield and sugar conversion rate of citric acid and shortening the fermentation period.

Drawings

FIG. 1 is an electrophoretogram of the Aspergillus Niger ATCC12846 genome;

FIG. 2 is a PAN7-1 plasmid map;

FIG. 3 is a PCR electrophoresis of upstream and downstream homology arms of the VosA gene, wherein M is DL5000 DNAmarker, lane 1 is the upper homology arm of VosA, lane 3 is the lower homology arm of VosA, and lane 5 is the hyg resistance expression element;

FIG. 4 is an electrophoretogram of a knockout fragment, wherein M is a marker lane and 1 is a knockout fragment;

FIG. 5 is a graph of crystal violet staining;

FIG. 6 is a graph showing the difference in OD values of crystal violet stains;

FIG. 7 shows the results of fermentation of original A.niger and A.niger genetically engineered bacteria.

FIG. 8 shows the difference between the original Aspergillus niger and Aspergillus niger genetically engineered bacteria 8 after continuous fermentation in batches

FIG. 9 is an electron micrograph of biofilm from original Aspergillus niger;

FIG. 10 is the electron microscope image of the biological membrane of Aspergillus niger genetically engineered bacteria.

Detailed Description

Example 1 construction of the Aspergillus niger Glucan Synthesis regulatory Gene VosA knockdown bacterium

Extraction of original Aspergillus niger genome

A kit for extracting a plant genome (takara minitest plant genomic DNA extraction kit) by the takara corporation is used, and the specific method is as follows:

1. inoculating 1mL of the scraped Aspergillus niger spore liquid into 50mL of DP culture medium, and culturing at 35 ℃ at 200r/min for 24 h; the DP medium formulation is as follows: 10g/L dextrin, 5g/L peptone, 2.5g/L potassium dihydrogen phosphate, 1g/L sodium nitrate, 0.5g/L magnesium sulfate and 10g/L glycine, and the volume is determined to be 50mL after water is added to 100 mL. (ii) a

2. Centrifuging at 8000r/min for 5min, collecting mycelium pellets, washing with normal saline twice, grinding the collected mycelium pellets with liquid nitrogen for 3 times, weighing 100mg of ground powder, adding into a tube with 500 μ L Buffer HS II, mixing, adding 10 μ L RNase A, shaking fully, mixing, and water-bathing at 56 deg.C for 10 min;

3. add 62.5. mu.L Buffer KAC to step 2 and mix well. The mixture was kept on ice for 5min and centrifuged at 12000rpm for 5 min. Taking 600 mu L of supernatant, adding 600 mu L of Buffer GB, and fully and uniformly mixing;

4. placing Spin Column in Collection Tube, transferring the solution into Spin Column, centrifuging at 12000rpm for 1min, and removing the filtrate;

5. adding 500 μ L Buffer WA into Spin Column, centrifuging at 12000rpm for 1min, and discarding the filtrate;

6. adding 700 mu L of Buffer WB into Spin Column, centrifuging at 12000rpm for 1min, and removing the filtrate;

7. repeating the step 6 once;

8. spin Column was mounted on the Collection Tube and centrifuged at 12000rpm for 2 min;

9. spin Column was placed in a new 1.5mL centrifuge tube, 40. mu.L of sterilized 65 ℃ water was added to the center of the Spin Column membrane, and the mixture was allowed to stand at room temperature for 1 min. The DNA was eluted by centrifugation at 12000rpm for 2 min. The Aspergillus niger genome concentration was determined by agarose gel electrophoresis as shown in FIG. 1.

FIG. 1 shows DNA marker with M DL15000 and the extracted A.niger genome No. 1.

(II) amplifying upstream and downstream homologous arms of gene VosA by using PCR technology

Amplifying an upstream homologous arm by using the original Aspergillus niger genome extracted in the step (I) as a template, using VosA-up-F as an upper primer and using VosA-up-R as a lower primer (the nucleotide sequence of the VosA-up-F is shown as SEQ ID NO:3, and the nucleotide sequence of the VosA-up-R is shown as SEQ ID NO: 4); the downstream homology arms were amplified using VosA-down-F as the upper primer and VosA-down-R as the lower primer (nucleotide sequence of VosA-down-F is shown in SEQ ID NO:5, and nucleotide sequence of VosA-down-R is shown in SEQ ID NO: 6). The reaction system is shown in Table 1, and the PCR reaction conditions are as follows: (1) denaturation at 98 ℃ for 10 s; (2) annealing at 55-65 ℃ for 30 s; (3) extending at 68 ℃ for 2min, and repeating the steps (1) to (3) for 30 times.

TABLE 1 PCR reaction System and reaction conditions

After the reaction was completed, the PCR product was quantified by agarose gel electrophoresis, as shown in FIG. 3. The nucleotide sequence of the upstream homologous arm of the gene VosA is shown as SEQ ID NO. 11, and the nucleotide sequence of the downstream homologous arm of the gene VosA is shown as SEQ ID NO. 12.

(III) amplification of hyg-resistant expression elements

Amplifying the hyg-resistant expression element by using PAN7-1 plasmid (Youbao, PAN7-1 plasmid map shown in figure 2, nucleotide sequence shown in SEQ ID NO:17) as a template, using VosA-hyg-F as an upper primer and using VosA-hyg-R as a lower primer (the nucleotide sequence of VosA-hyg-F is shown in SEQ ID NO:7 and the nucleotide sequence of VosA-hyg-R is shown in SEQ ID NO: 8). The reaction system is shown in Table 1, and the PCR reaction conditions are as follows: (1) denaturation at 98 ℃ for 10 s; (2) annealing at 55-65 ℃ for 30 s; (3) extending at 68 ℃ for 3min, and repeating the steps (1) to (3) for 30 times. After the reaction was completed, the PCR product was quantified by agarose gel electrophoresis, as shown in FIG. 3. Wherein the nucleotide sequence of the hyg resistance expression element is shown as SEQ ID NO. 13.

FIG. 3 shows DNA Marker with M being DL5000, upper homology arm with No. 1 being VosA, lower homology arm with No. 3 being VosA, and hyg resistance expression element with No. 5.

(IV) amplification of knockout fragment

The VosA upstream homologous arm, the downstream homologous arm and the hyg resistance expression element are used as templates, VosA-F is used as an upper primer, VosA-R is used as a lower primer (the nucleotide sequence of VosA-F is shown as SEQ ID NO:9, and the nucleotide sequence of VosA-R is shown as SEQ ID NO: 10), and the VosA gene knockout fragment is amplified by using overlap extension PCR (overlap PCR) technology. The reaction system is shown in Table 1, and the PCR reaction conditions are as follows: (1) denaturation at 98 ℃ for 10 s; (2) annealing at 55-65 ℃ for 30 s; (3) extending at 68 ℃ for 7min, and repeating the steps (1) to (3) for 30 times. After completion of the reaction, the PCR product was quantified by agarose gel electrophoresis, as shown in FIG. 4. The nucleotide sequence of the gene knockout fragment is shown as SEQ ID NO. 14.

FIG. 4 shows DNA Marker with M being DL10000 and No. 1 being a knockout fragment.

(V) preparation and transformation of Aspergillus niger protoplast

1. Aspergillus niger was inoculated into PDA plates and 3mL of the sporulation buffer was added to the plates after spores had grown, the spores were scraped off with a coating stick and transferred to a sterilized 5mL centrifuge tube.

The culture medium formula of the PDA plate is as follows: self-making, weighing about 200g of potato, peeling, and cutting into 1cm pieces³Adding 600mL water into small pieces, boiling for 30min, filtering with 4 layers of gauze to obtain potato juice, metering to 1L, and packaging into 5 bottles of 200 mL. Then 20g/L glucose and 15g/L agar powder are added, and the mixture is sterilized for 20min at 115 ℃.

2. Inoculating 0.5mL spore solution to 50mL DP medium, culturing at 35 deg.C and 200rpm for 13-16 h.

3. After the spores were germinated, they were filtered through Miracloth (Miracloth) to leave hyphae.

Preparing enzymolysis solution (0.1 g/10mL each of lyase, collapsing enzyme, snailase, 400 μ L/10mL of cellulase), and filtering and sterilizing with sterile syringe.

4. Adding 2g of mycelium into the enzyme solution, and performing enzymolysis at 30 ℃ and 220rpm for 30 min; then the rotating speed is reduced to 150rpm for enzymolysis for 3 h.

5. After the enzymolysis is finished, filtering with filter paper, taking the filtrate, and centrifuging at 5000rpm for 10min at 4 ℃. Removing supernatant, adding 1mL of 1M sorbitol aqueous solution (ice water bath), uniformly mixing by blowing and sucking with a gun, adding 15mL of 1M sorbitol aqueous solution, centrifuging, and removing supernatant. And then repeated once more. The supernatant was removed, 1mL Solution5 was added, and the mixture was mixed by gun-suction to obtain protoplasts. Wherein, the Solution5 is self-made, and the formula is as follows: KCl 4.47g, CaCl₂0.735g, MOP 0.2093g and KOH to adjust the pH to 6.0, and the volume is 100mL by water.

6. And (3) sucking 100 mu L of protoplast into a 1.5mL sterile centrifuge tube, and adding 10 mu L of the gene knockout fragment constructed in the step (IV) to the protoplast and mixing the fragment and the protoplast. Then adding 50 mu L of Solution4, mixing uniformly, placing on ice and timing for 15-30 min. Wherein, the Solution4 is self-made, and the formula is as follows: PEG 800025 g, CaCl21.47g, KCl 4.47g, 10mM Tris, pH adjusted to 7.5 with hydrochloric acid, and water to 100 mL.

After 7.20min, 900 μ L of Solution4 was added, turned upside down several times, left at room temperature and timed for 15-30 min. And (3) after 15-30min, centrifuging at 6000rpm for 5min, discarding 900 mu L of supernatant, coating the residual thallus in a PDA culture medium with sucrose concentration of 1mol/L and hygromycin concentration of 60mmol/L, and performing upright culture to obtain a transformant.

8. Transformants were picked for colony PCR validation. The specific method comprises the following steps: an appropriate amount of the transformant was added to 50. mu.L of colony PCR buffer (100mM/L Tris-HCl, 10mM/L EDTA, 1M/L KCl), and 0.5. mu.L was added to the PCR reaction system in a water bath at 95 ℃ for 10 min. PCR primers are hyg-F and hyg-R (the nucleotide sequence of hyg-F is shown as SEQ ID NO:15, and the nucleotide sequence of hyg-R is shown as SEQ ID NO: 16), agarose electrophoresis shows an amplification band, which indicates that the transformation is successful, and a delta VosA strain is obtained.

Example 2 Crystal Violet staining experiment

Respectively inoculating original Aspergillus Niger (Aspergillus Niger ATCC 12846) and genetically engineered bacteria to a PDA (personal digital assistant) plate, adding 3mL of a sporulation buffer solution into the plate when spores grow, scraping the spores by using a coating rod, transferring the spores into a sterilized 5mL centrifuge tube, and fixing the volume to 2mL by using the sporulation buffer solution to obtain a spore solution. Quantitated and diluted to 10 using a hemocytometer⁶one/mL, followed by further dilution to 10⁵/mL，10⁴/mL。

1mL of synthetic medium was added to a 24-well plate in advance, and then 2. mu.L of spore liquid at different concentrations was inoculated into the medium. Standing and culturing at 35 ℃ for 36h to enable Aspergillus niger to form a film at the bottom of the pore plate. The medium was then decanted, washed 2 times with PBS, and stained with 0.1% crystal violet for 15 min. Then pouring out the crystal violet, washing with PBS for 2 times, adding glacial acetic acid, and placing in a shaking instrument for 30min to decolorize the crystal violet. Then, observation and detection of OD570 by microplate reader were carried out. The crystal violet staining pattern and the difference in OD value are shown in FIGS. 5 and 6.

The formula of the synthetic culture medium is as follows: 6g/L of sodium nitrate, 0.52g/L of potassium chloride, 0.52g/L of magnesium sulfate, 1.52g/L of monopotassium phosphate, 10g/L of glucose and 0.4mg/L of biotin.

TABLE 2 OD values of biofilm crystal violet staining experiments at different spore concentrations

The results in FIGS. 5 and 6 show that the Δ VosA strain, after decolourisation, is markedly lighter in purple colour than the original strain, at 10³The color of the delta VosA strain at the concentration is completely removed, and the data result is consistent with the color according to the OD value detected by an enzyme-labeling instrument. Shows that the biomembrane is reduced after the inactivation of the glucan synthesis regulatory gene VosA.

Example 3 immobilized fermentation experiment of genetically engineered bacteria

1. Preparation of porous fiber material immobilized medium

Soaking porous fiber material (aperture 1mm) in anhydrous ethanol for 1 hr, washing with water until no ethanol smell is observed, and oven drying at 65 deg.C to constant weight. Cutting into 0.5cm³The vectors with the same size are arranged on the left and the right.

2. Preparation of fermentation Medium

Weighing 250g/L of corn flour, placing the corn flour in a 75 ℃ water bath, adding 1mL of liquefying enzyme (60000U/L) when the corn flour liquid reaches 65 ℃, liquefying for 40min, then heating the water bath to 95 ℃, adding 1mL of liquefying enzyme when the corn flour liquid reaches 85 ℃, liquefying for 60min until the iodine solution does not turn blue, filtering the corn flour liquid to obtain corn flour clear liquid, adding the unfiltered corn flour liquid as a return material (the volume ratio of the unfiltered corn flour liquid to the corn clear liquid is 8 percent), and uniformly mixing. Each 100mL of the suspension was dispensed into 500mL Erlenmeyer flasks containing 2g/L of the carrier. Sterilizing and cooling for later use.

3. Fermentation of

(1) Inoculating the frozen Aspergillus niger genetically engineered bacteria and original Aspergillus niger spores onto a PDA (personal digital assistant) plate, and culturing in a constant-temperature incubator at 35 ℃ for 4-5 days until the plate is full of spores.

(2) Scraping spores with a spore scraping buffer solution to obtain a spore suspension, taking a proper amount of the spore suspension, transferring the spore suspension into a 500mL conical flask filled with 100mL of immobilized culture medium, culturing for 96h at 35 ℃ in a shaking table at 250rpm, sampling every 12h in the fermentation process, centrifuging for 5min at 12000rpm, separating the supernatant from the precipitate, and measuring the residual sugar concentration and the citric acid yield in the supernatant. When the concentration of residual sugar is lower than 5g/L, the fermentation is finished. Wherein the citric acid is determined by NaOH titration method and the total sugar is determined by DNS method.

Wherein the NaOH titration method comprises the following steps: 1mL of sample (diluted to a certain concentration) is added into a 250mL conical flask, 50mL of pure water is added at the same time, 0.1429M NaOH is used for titration, and the consumed amount of NaOH is the yield of the citric acid.

The DNS method comprises the following steps:

and (3) DNS preparation: weighing 10g of 3, 5-dinitrosalicylic acid, placing the 3, 5-dinitrosalicylic acid in about 600mL of water, gradually adding 10g of sodium hydroxide, magnetically stirring the mixture in a water bath at 50 ℃ to dissolve the sodium hydroxide, then sequentially adding 200g of sodium methyl tartrate, 2g of phenol and 5g of anhydrous sodium sulfite, cooling the mixture to room temperature after the sodium methyl tartrate, the phenol and the anhydrous sodium sulfite are completely dissolved and clarified, and fixing the volume to 1000mL by using pure water. Stored in a brown reagent bottle and used after being placed in the dark for 7 days.

The preparation method of the standard yeast comprises the following steps: preparing a series of sugar standard solutions with the concentration of 0.0-1.0g/L, respectively adding 0.5mL of the sugar standard solutions into 15mL centrifuge tubes at each concentration, then adding 0.5mL of DNS solution into each centrifuge tube,

placing the centrifuge tubes in a boiling water bath for reaction for 5min, placing the centrifuge tubes in ice water for cooling, then adding 8mL of pure water into each centrifuge tube, uniformly mixing, measuring the light absorption value OD540 under the wavelength of 540nm, and taking 0.0g/L as a control. The concentration of the standard solution is ordinate and the light absorption value OD₅₄₀A standard curve is plotted for the abscissa.

The sample measuring method comprises the following steps: after the sample is diluted properly, 1mL of concentrated sulfuric acid is added into 10mL of the sample, the mixture is placed into ice water for cooling after 15min of reaction in a boiling water bath, the pH value is adjusted to be neutral, the volume is determined to be 100mL, then the mixture is diluted to the proper concentration, and the measurement method is the same as the above. Different sugars correspond to different standards.

FIG. 7 shows the difference between the Δ VosA strain and the original strain under fermentation conditions, and the results show that after 96h fermentation, the yield of citric acid produced by fermentation of Δ VosA strain is 172.1g/L on average, and the yield of citric acid produced by fermentation of the original strain is 160.4g/L on average. In the conversion rate, the conversion rate of the delta VosA strain reaches 98.9%, the conversion rate is improved by 6.6% compared with that of the original strain, and the period is also reduced from 84h to 72 h. FIG. 8 shows the difference in production intensity of the Δ VosA strain and the original strain after 8 consecutive fermentations. As can be seen from the figure, the fermentation intensity of the strain Δ VosA is not significantly reduced after 8 batches of continuous fermentation, but is reduced by 41.4% after 8 batches of the original strain, which shows that the continuous fermentation performance of the strain Δ VosA is significantly better than that of the original strain. Aspergillus Niger ATCC12846 is a high-yield citric acid strain, on the basis, the yield can be improved, the period is obviously shortened, the continuous fermentation effect is also obviously enhanced, so that the Aspergillus Niger immobilized fermentation method is greatly improved on the basis of producing citric acid by Aspergillus Niger immobilized fermentation, and is suitable for industrial production.

Example 4 SEM Observation of biofilms

The immobilized carrier after fermentation for 72h was taken out, washed 3 times with PBS to remove adsorbed mycelia. The mixture was placed in a freezer at-80 ℃ overnight and then lyophilized in a lyophilizer. And the carrier is adhered on a point mirror table through conductive glue, and 20mA 30s is subjected to gold spraying. Then, the sample was taken into TM3000 for observation. The electron micrographs of the biofilm are shown in FIGS. 9 and 10. FIG. 9 shows immobilization of the A.niger original strain, and FIG. 10 shows immobilization of the. DELTA.VosA strain. It was found that the Aspergillus niger original bacteria formed a plurality of spheres on the carrier, and these spheres connected to form a film and simultaneously blocked the pore size of the carrier, which affected the oxygen and mass transfer of the inner bacteria. And the delta VosA strain forms a small amount of biomembrane at the corners and on the surface of the carrier, does not influence oxygen and mass transfer of internal thalli, and is favorable for producing citric acid by immobilized fermentation.

Sequence listing

<110> Nanjing high and new Industrial and Biotechnology research institute Co., Ltd

<120> Aspergillus niger genetically engineered bacterium and construction method and application thereof

<160>17

<170>SIPOSequenceListing 1.0

<210>1

<211>2131

<212>DNA

<213> Aspergillus niger (Aspergillus niger)

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aactgactat gattaccagc tccgactttg acctgatcgt tcgccagcag cccaacaggg 180

cgcgcgtggc gggtgggaag gagaaaggta tgtggaatca ccggctttgc tagtctcttc 240

ggtttttctt cctgagaagc aacgccccta cggacgagca gaggttgact tgggacccgt 300

tttcgcgctg cagaacgcaa gccagtagat ccgccgccga ttgtgcagat ccgagtcaga 360

gaggagggga cctatctcgc gcagtaggtg ttcgcgcttg aaattggtgc tgcgttggag 420

aaacaggccc actgacaaaa caccgatagg cattacttgc aaagccctta ttattttatg 480

tgctgcagct tgtatgatgc gtcagaggat aacccagttc ccgtggcccc gtccactgct 540

ctgaccggca cactggtttc gtctctccac aggttgaaag atgtggacaa tactggtaag 600

tttggtcttc gaatgagctt caaaaacagg tctagatttg gcttactgac tggacctggc 660

ccagatgggg gattcttcgt ttggggggac ttgtcagtaa aaatcgaagg agatttccgg 720

ctcaagttca ctctcttcga aatgcgaaag taggtttctt tggccctgca atggctccca 780

gcgagcctct ccacagactt gtgagaagcg catgctgatt ttgcggcgat ttaaagggac 840

gtcgtaaccc atcttaagtc catcatcacg gatagattta ccggttcgtt tacgtcctcg 900

tgaaatcctt ttcttgagaa cgcagctcct gactccgtac agtgtacccg ccaaagagct 960

ttccgggcat ggcagagtcg acgttcctct ccagatcttt tgcggaccaa ggtgtgaagt 1020

tgagaattag aaaggagcca cgaacactaa tgtgagtgaa gtagccgagt gactgggaaa 1080

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gcaacgcgtt cagcgcttac ccgacaacga gtagagacta tgggtactat ggacagcaga 1260

cacaccccag taagcggccg cgaatgtcgt ctatagacct tgggacccga gggatgtatg 1320

acgcagatgg gcggatgcgt cagatggata cgtaccctca aacggcgatc tacaatcagc 1380

cagggggcta cgcaactcct atgatgcaag gctatcctgc cggacacacg gcggttccag 1440

attatgcggt tcgtctgcct gattcattac ttcaaagacc ggcttgggag gacagcaatg 1500

ctcccacgaa gcgccgtctc cagaggccgg atgtttctag caggatgcta acaatatagc 1560

agatgtctta cggaatccca tcctccactc aggtacccca gatgcaagac cctggggcac 1620

acggtcgatc cagccaacag gccacgatgc agtccttagg aatggtcaac gcgcctggta 1680

ctcctgtatg gactccgcct accacccgtc gttaaggtcg agaagctaag aaccgtatat 1740

tattctggat agacagcgga ctcgacggga gcaatgatgc cacaaagcta ccctcgttcg 1800

caataccaga ctgggtccac aatccttcct cctttgcaac aacggaatcg aaattttgct 1860

caaggcacca acggtgcagc ggcacgaggc tactttgatc aatcttccca agcggcgaca 1920

ccgatccttc catcacaacc tattcctacc aatgaagtgg acaggtacgg ttctactcct 1980

ggccaggcgt cttttgagca tcctgcttca gcgaatggga ccccgcgatg agtggtttcg 2040

gagtgctcag ccactgtggc tcggttgctt gtggtcttga tcgatggtcg aatttgctct 2100

ggttgttctt tctctctctc tctttgcttt t 2131

<210>2

<211>1021

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

agtgagacag caaacggcct gtaccccggc cagaggagta tccgcagccg attccgcgct 60

cgcccgatcg ttcttcgatc cagatgccaa gcaacgcgtt cagcgcttac ccgacaacga 120

gtagagacta tgggtactat ggacagcaga cacaccccag taagcggccg cgaatgtcgt 180

ctatagacct tgggacccga gggatgtatg acgcagatgg gcggatgcgt cagatggata 240

cgtaccctca aacggcgatc tacaatcagc cagggggcta cgcaactcct atgatgcaag 300

gctatcctgc cggacacacg gcggttccag attatgcggt tcgtctgcct gattcattac 360

ttcaaagacc ggcttgggag gacagcaatg ctcccacgaa gcgccgtctc cagaggccgg 420

atgtttctag caggatgcta acaatatagc agatgtctta cggaatccca tcctccactc 480

aggtacccca gatgcaagac cctggggcac acggtcgatc cagccaacag gccacgatgc 540

agtccttagg aatggtcaac gcgcctggta ctcctgtatg gactccgcct accacccgtc 600

gttaaggtcg agaagctaag aaccgtatat tattctggat agacagcgga ctcgacggga 660

gcaatgatgc cacaaagcta ccctcgttcg caataccaga ctgggtccac aatccttcct 720

cctttgcaac aacggaatcg aaattttgct caaggcacca acggtgcagc ggcacgaggc 780

tactttgatc aatcttccca agcggcgaca ccgatccttc catcacaacc tattcctacc 840

aatgaagtgg acaggtacgg ttctactcct ggccaggcgt cttttgagca tcctgcttca 900

gcgaatggga ccccgcgatg agtggtttcg gagtgctcag ccactgtggc tcggttgctt 960

gtggtcttga tcgatggtcg aatttgctct ggttgttctt tctctctctc tctttgcttt 1020

t 1021

<210>3

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

ttcagtcttg cgcaggtggc 20

<210>4

<211>40

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

ggtggaggcg gcggatttta aggacagaca gctgcgaacc 40

<210>5

<211>42

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

cccactccac atctccactc gaagtgagac agcaaacggc ct 42

<210>6

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

aggctgaagc cgaaccaact 20

<210>7

<211>40

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

ggttcgcagc tgtctgtcct taaaatccgc cgcctccacc 40

<210>8

<211>42

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

aggccgtttg ctgtctcact tcgagtggag atgtggagtg gg 42

<210>9

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

tggttcaccc ttcagctggc 20

<210>10

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

cctctggaga agcctggagc20

<210>11

<211>1806

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

ttcagtcttg cgcaggtggc cgcagtttgg atgcgacttt ttgtcatttc ccctccctcg 60

ctttttttcc tcgggacttg ccaatgtgcg catcatctcc atgtcgctgc ggttcctgtc 120

ggatcgtcaa ggctgataca acaccctagc ccgcatgccc aaccgtggtt gacctgactg 180

ggaacgatac cattcgacgg ccgaacagtg gccggcgttg ctctgcgatt tccggtacag 240

ctccactagg ccatttgtct ggttcaccct tcagctggct gtcctcttga ttaccagtaa 300

atttttaccc tagacttcct aattttgaca caccacgggc accacgcatt tcttttcgtg 360

gatttttgtg cgtcagagac ctgtcagagg cgactttgtt gacccatcgt tcagctaccg 420

tctgatcgct gggcggtgtt caatcccgcc acacctagtg ctcgctcgct tcaaactact 480

attgctgctg ctgctactat tattattcat ttgacttttg ggcaaatttt gttttgaagg 540

aggggcgcga aaattatttt gttttccgct tgagttggtc tgtgatttcc ctcaattatt 600

attttttttt tctctcttta ctcggccgcg cgatctgtga tgtctagacc cgggcgtcca 660

tttgaatgac ccaaacacca cggtcgcagc agagcaccat ttggattccc ggaattttct 720

tcaggtgaag gtgccaaaag aaagattggc caggcaccca cgacgtttca tcctcacgtg 780

aagaatatcc ctccaagggt gttgaaaaaa aatattactg ggaagacttc gaaagagcca 840

aagggtgaag ggaaaaaaat tttttttttt tttctttaag ggaaaggaag tctttgaata 900

acacgcaaaa acaagtgaaa taaaacaaga ccgacacata aagagagggg aggaaaaaaa 960

aaaaaaagga agcgaaaaaa gagaaaacca aaagagttta attggagaaa gaaaaaagga 1020

aaaagaaaaa agaaaaaaaa aagaaagaaa attaacaaga ggaaaataaa gaaaagttga 1080

cgaacacgct tctactacat tattttcgaa tcagagccgc agcaacaaca gcaacagcag 1140

cagcagcagc cgcaaaaccc aagcagcttt actcccctat aagagccaaa agaaagcagc 1200

ccagtcacaa gtgagaagag gtggaaaagg aagcaaagca acttagttag caatccactt 1260

gggtcgaatt ggctggttaa ggtttgtctg gaaccgtgct cggctggctc ttgccgcttg 1320

cattctgacg acaccatcca tctttttctt ccttctcggc gccgttttcg taccgcattc 1380

gcctcattgc agctcaaaaa ctacttgacg gtgctactgc aatttcggcc atccttccca 1440

gcgtcccgac gcaagtggca gcccagacgg tgttttccta cggattctcc actccggaag 1500

ttgggtcgaa ggtccagctc cagctgctac ggactgacta actagtcaat cataccgtcg 1560

tgtcccatct ttaaccaacc ccaactcccc tctttcaccc ccaccgtgta tcattctctc 1620

ttctagtgct tcgtcgaacc acaagtcgct ggctgctagg ttccagagac ccgaactctt 1680

cttccctgct gttaatccat cggttcattc tttgtttctt catcatatct cacttttccc 1740

ctggccacct tctgttgttc agacgtggtt cgcagctgtc tgtccttaaa atccgccgcc 1800

tccacc 1806

<210>12

<211>1972

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

cccactccac atctccactc gaagtgagac agcaaacggc ctgtaccccg gccagaggag 60

tatccgcagc cgattccgcg ctcgcccgat cgttcttcga tccagatgcc aagcaacgcg 120

ttcagcgctt acccgacaac gagtagagac tatgggtact atggacagca gacacacccc 180

agtaagcggc cgcgaatgtc gtctatagac cttgggaccc gagggatgta tgacgcagat 240

gggcggatgc gtcagatgga tacgtaccct caaacggcga tctacaatca gccagggggc 300

tacgcaactc ctatgatgca aggctatcct gccggacaca cggcggttcc agattatgcg 360

gttcgtctgc ctgattcatt acttcaaaga ccggcttggg aggacagcaa tgctcccacg 420

aagcgccgtc tccagaggcc ggatgtttct agcaggatgc taacaatata gcagatgtct 480

tacggaatcc catcctccac tcaggtaccc cagatgcaag accctggggc acacggtcga 540

tccagccaac aggccacgat gcagtcctta ggaatggtca acgcgcctgg tactcctgta 600

tggactccgc ctaccacccg tcgttaaggt cgagaagcta agaaccgtat attattctgg 660

atagacagcg gactcgacgg gagcaatgat gccacaaagc taccctcgtt cgcaatacca 720

gactgggtcc acaatccttc ctcctttgca acaacggaat cgaaattttg ctcaaggcac 780

caacggtgca gcggcacgag gctactttga tcaatcttcc caagcggcga caccgatcct 840

tccatcacaa cctattccta ccaatgaagt ggacaggtac ggttctactc ctggccaggc 900

gtcttttgag catcctgctt cagcgaatgg gaccccgcga tgagtggttt cggagtgctc 960

agccactgtg gctcggttgc ttgtggtctt gatcgatggt cgaatttgct ctggttgttc 1020

tttctctctc tctctttgct tttacctcct tggatagaca gttggacggg tgttttgctt 1080

ttactgggaa caagaatcgg gctctgggga taaatggcgc ctcggggatt taaaatggga 1140

taaccatact acaggtgttt gattgttatt tctattcccc gttattatgt tatggttacg 1200

gctttggatc gagtgatgac ggatgatggc tggacggttg acttcattgt acatattaac 1260

ttgaaacaga atttgacatg agatgggtag cttaatggct atgctgccac ttctcgttat 1320

tattaagttg aactagcccg aagcaaatca actggtgttt gtaaggttcc ttccagcacc 1380

gaaactatat acgcggacta aggctggctg cctgaggtta ggccggatgt tgccttcctt 1440

tttgggggaa aatcccaact ggaggtggga ttcaaccggc ttcttcccct cgttgacagc 1500

ctcgccttca cttctcactc cacagttgct agcattcact ctttcggcct tgctccccac 1560

tttgctctta cgatcatttt gcctacacgc agctgtgttg tctctgttgt cgtccatccg 1620

cgtctgctct gatattctta ccccaacgca tcgtcaaacc accactgtcc taatcatggc 1680

ttcctacaaa gacggaagcc gtcaagtacc tctgctccag gcttctccag aggtttcaaa 1740

tttggacatt cgaagtacct ggagcggtag cctagacgag cccgtcaact gcagaatctc 1800

tatggtggtg agctgctaca tccccattac tgtactctgt ccgctgctca tcgagaattt 1860

ctccccttct tcggactagg tccattgtat tccgagcacg cagctatcta tcttgaacat 1920

ccgatctgcc ggggtttcta tcgtcaatac acagttggtt cggcttcagc ct 1972

<210>13

<211>2780

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

ggttcgcagc tgtctgtcct taaaatccgc cgcctccacc atttgtagaa aaatgtgacg 60

aactcgtgag ctctgtacag tgaccggtga ctctttctgg catgcggaga gacggacgga 120

cgcagagaga agggctgagt aataagccac tggccagaca gctctggcgg ctctgaggtg 180

cagtggatga ttattaatcc gggaccggcc gcccctccgc cccgaagtgg aaaggctggt 240

gtgcccctcg ttgaccaaga atctattgca tcatcggaga atatggagct tcatcgaatc 300

accggcagta agcgaaggag aatgtgaagc caggggtgta tagccgtcgg cgaaatagca 360

tgccattaac ctaggtacag aagtccaatt gcttccgatc tggtaaaaga ttcacgagat 420

agtaccttct ccgaagtagg tagagcgagt acccggcgcg taagctccct aattggccca 480

tccggcatct gtagggcgtc caaatatcgt gcctctcctg ctttgcccgg tgtatgaaac 540

cggaaaggcc gctcaggagc tggccagcgg cgcagaccgg gaacacaagc tggcagtcga 600

cccatccggt gctctgcact cgacctgctg aggtccctca gtccctggta ggcagctttg 660

ccccgtctgt ccgcccggtg tgtcggcggg gttgacaagg tcgttgcgtc agtccaacat 720

ttgttgccat attttcctgc tctccccacc agctgctctt ttcttttctc tttcttttcc 780

catcttcagt atattcatct tcccatccaa gaacctttat ttcccctaag taagtacttt 840

gctacatcca tactccatcc ttcccatccc ttattccttt gaacctttca gttcgagctt 900

tcccacttca tcgcagcttg actaacagct accccgcttg agcagacatc accatgcctg 960

aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc 1020

tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg 1080

gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc 1140

ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg 1200

agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg 1260

aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg 1320

ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca 1380

ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg 1440

tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg 1500

ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc 1560

tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt 1620

cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc 1680

agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt 1740

atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg 1800

atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg 1860

ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac 1920

tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa tagagtagat 1980

gccgaccgcg ggatccactt aacgttactg aaatcatcaa acagcttgac gaatctggat 2040

ataagatcgt tggtgtcgat gtcagctccg gagttgagac aaatggtgtt caggatctcg 2100

ataagatacg ttcatttgtc caagcagcaa agagtgcctt ctagtgattt aatagctcca 2160

tgtcaacaag aataaaacgc gttttcgggt ttacctcttc cagatacagc tcatctgcaa 2220

tgcattaatg cattgactgc aacctagtaa cgccttncag gctccggcga agagaagaat 2280

agcttagcag agctattttc attttcggga gacgagatca agcagatcaa cggtcgtcaa 2340

gagacctacg agactgagga atccgctctt ggctccacgc gactatatat ttgtctctaa 2400

ttgtactttg acatgctcct cttctttact ctgatagctt gactatgaaa attccgtcac 2460

cagcncctgg gttcgcaaag ataattgcat gtttcttcct tgaactctca agcctacagg 2520

acacacattc atcgtaggta taaacctcga aatcanttcc tactaagatg gtatacaata 2580

gtaaccatgc atggttgcct agtgaatgct ccgtaacacc caatacgccg gccgaaactt 2640

ttttacaact ctcctatgag tcgtttaccc agaatgcaca ggtacacttg tttagaggta 2700

atccttcttt ctagaagtcc tcgtgtactg tgtaagcgcc cactccacat ctccactcga 2760

agtgagacag caaacggcct 2780

<210>14

<211>5978

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

tggttcaccc ttcagctggc tgtcctcttg attaccagta aatttttacc ctagacttcc 60

taattttgac acaccacggg caccacgcat ttcttttcgt ggatttttgt gcgtcagaga 120

cctgtcagag gcgactttgt tgacccatcg ttcagctacc gtctgatcgc tgggcggtgt 180

tcaatcccgc cacacctagt gctcgctcgc ttcaaactac tattgctgct gctgctacta 240

ttattattca tttgactttt gggcaaattt tgttttgaag gaggggcgcg aaaattattt 300

tgttttccgc ttgagttggt ctgtgatttc cctcaattat tatttttttt ttctctcttt 360

actcggccgc gcgatctgtg atgtctagac ccgggcgtcc atttgaatga cccaaacacc 420

acggtcgcag cagagcacca tttggattcc cggaattttc ttcaggtgaa ggtgccaaaa 480

gaaagattgg ccaggcaccc acgacgtttc atcctcacgt gaagaatatc cctccaaggg 540

tgttgaaaaa aaatattact gggaagactt cgaaagagcc aaagggtgaa gggaaaaaaa 600

tttttttttt ttttctttaa gggaaaggaa gtctttgaat aacacgcaaa aacaagtgaa 660

ataaaacaag accgacacat aaagagaggg gaggaaaaaa aaaaaaaagg aagcgaaaaa 720

agagaaaacc aaaagagttt aattggagaa agaaaaaagg aaaaagaaaa aagaaaaaaa 780

aaagaaagaa aattaacaag aggaaaataa agaaaagttg acgaacacgc ttctactaca 840

ttattttcga atcagagccg cagcaacaac agcaacagca gcagcagcag ccgcaaaacc 900

caagcagctt tactccccta taagagccaa aagaaagcag cccagtcaca agtgagaaga 960

ggtggaaaag gaagcaaagc aacttagtta gcaatccact tgggtcgaat tggctggtta 1020

aggtttgtct ggaaccgtgc tcggctggct cttgccgctt gcattctgac gacaccatcc 1080

atctttttct tccttctcgg cgccgttttc gtaccgcatt cgcctcattg cagctcaaaa 1140

actacttgac ggtgctactg caatttcggc catccttccc agcgtcccga cgcaagtggc 1200

agcccagacg gtgttttcct acggattctc cactccggaa gttgggtcga aggtccagct 1260

ccagctgcta cggactgact aactagtcaa tcataccgtc gtgtcccatc tttaaccaac 1320

cccaactccc ctctttcacc cccaccgtgt atcattctct cttctagtgc ttcgtcgaac 1380

cacaagtcgc tggctgctag gttccagaga cccgaactct tcttccctgc tgttaatcca 1440

tcggttcatt ctttgtttct tcatcatatc tcacttttcc cctggccacc ttctgttgtt 1500

cagacgtggt tcgcagctgt ctgtccttaa aatccgccgc ctccaccatt tgtagaaaaa 1560

tgtgacgaac tcgtgagctc tgtacagtga ccggtgactc tttctggcat gcggagagac 1620

ggacggacgc agagagaagg gctgagtaat aagccactgg ccagacagct ctggcggctc 1680

tgaggtgcag tggatgatta ttaatccggg accggccgcc cctccgcccc gaagtggaaa 1740

ggctggtgtg cccctcgttg accaagaatc tattgcatca tcggagaata tggagcttca 1800

tcgaatcacc ggcagtaagc gaaggagaat gtgaagccag gggtgtatag ccgtcggcga 1860

aatagcatgc cattaaccta ggtacagaag tccaattgct tccgatctgg taaaagattc 1920

acgagatagt accttctccg aagtaggtag agcgagtacc cggcgcgtaa gctccctaat 1980

tggcccatcc ggcatctgta gggcgtccaa atatcgtgcc tctcctgctt tgcccggtgt 2040

atgaaaccgg aaaggccgct caggagctgg ccagcggcgc agaccgggaa cacaagctgg 2100

cagtcgaccc atccggtgct ctgcactcga cctgctgagg tccctcagtc cctggtaggc 2160

agctttgccc cgtctgtccg cccggtgtgt cggcggggtt gacaaggtcg ttgcgtcagt 2220

ccaacatttg ttgccatatt ttcctgctct ccccaccagc tgctcttttc ttttctcttt 2280

cttttcccat cttcagtata ttcatcttcc catccaagaa cctttatttc ccctaagtaa 2340

gtactttgct acatccatac tccatccttc ccatccctta ttcctttgaa cctttcagtt 2400

cgagctttcc cacttcatcg cagcttgact aacagctacc ccgcttgagc agacatcacc 2460

atgcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc 2520

tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga 2580

gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat 2640

gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa 2700

ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac 2760

ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc 2820

gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt 2880

caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg 2940

caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg 3000

ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac 3060

aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc 3120

ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg 3180

gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc 3240

cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat 3300

ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg 3360

actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta 3420

gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatag 3480

agtagatgcc gaccgcggga tccacttaac gttactgaaa tcatcaaaca gcttgacgaa 3540

tctggatata agatcgttgg tgtcgatgtc agctccggag ttgagacaaa tggtgttcag 3600

gatctcgata agatacgttc atttgtccaa gcagcaaaga gtgccttcta gtgatttaat 3660

agctccatgt caacaagaat aaaacgcgtt ttcgggttta cctcttccag atacagctca 3720

tctgcaatgc attaatgcat tgactgcaac ctagtaacgc cttncaggct ccggcgaaga 3780

gaagaatagc ttagcagagc tattttcatt ttcgggagac gagatcaagc agatcaacgg 3840

tcgtcaagag acctacgaga ctgaggaatc cgctcttggc tccacgcgac tatatatttg 3900

tctctaattg tactttgaca tgctcctctt ctttactctg atagcttgac tatgaaaatt 3960

ccgtcaccag cncctgggtt cgcaaagata attgcatgtt tcttccttga actctcaagc 4020

ctacaggaca cacattcatc gtaggtataa acctcgaaat canttcctac taagatggta 4080

tacaatagta accatgcatg gttgcctagt gaatgctccg taacacccaa tacgccggcc 4140

gaaacttttt tacaactctc ctatgagtcg tttacccaga atgcacaggt acacttgttt 4200

agaggtaatc cttctttcta gaagtcctcg tgtactgtgt aagcgcccac tccacatctc 4260

cactcgaagt gagacagcaa acggcctgta ccccggccag aggagtatcc gcagccgatt 4320

ccgcgctcgc ccgatcgttc ttcgatccag atgccaagca acgcgttcag cgcttacccg 4380

acaacgagta gagactatgg gtactatgga cagcagacac accccagtaa gcggccgcga 4440

atgtcgtcta tagaccttgg gacccgaggg atgtatgacg cagatgggcg gatgcgtcag 4500

atggatacgt accctcaaac ggcgatctac aatcagccag ggggctacgc aactcctatg 4560

atgcaaggct atcctgccgg acacacggcg gttccagatt atgcggttcg tctgcctgat 4620

tcattacttc aaagaccggc ttgggaggac agcaatgctc ccacgaagcg ccgtctccag 4680

aggccggatg tttctagcag gatgctaaca atatagcaga tgtcttacgg aatcccatcc 4740

tccactcagg taccccagat gcaagaccct ggggcacacg gtcgatccag ccaacaggcc 4800

acgatgcagt ccttaggaat ggtcaacgcg cctggtactc ctgtatggac tccgcctacc 4860

acccgtcgtt aaggtcgaga agctaagaac cgtatattat tctggataga cagcggactc 4920

gacgggagca atgatgccac aaagctaccc tcgttcgcaa taccagactg ggtccacaat 4980

ccttcctcct ttgcaacaac ggaatcgaaa ttttgctcaa ggcaccaacg gtgcagcggc 5040

acgaggctac tttgatcaat cttcccaagc ggcgacaccg atccttccat cacaacctat 5100

tcctaccaat gaagtggaca ggtacggttc tactcctggc caggcgtctt ttgagcatcc 5160

tgcttcagcg aatgggaccc cgcgatgagt ggtttcggag tgctcagcca ctgtggctcg 5220

gttgcttgtg gtcttgatcg atggtcgaat ttgctctggt tgttctttct ctctctctct 5280

ttgcttttac ctccttggat agacagttgg acgggtgttt tgcttttact gggaacaaga 5340

atcgggctct ggggataaat ggcgcctcgg ggatttaaaa tgggataacc atactacagg 5400

tgtttgattg ttatttctat tccccgttat tatgttatgg ttacggcttt ggatcgagtg 5460

atgacggatg atggctggac ggttgacttc attgtacata ttaacttgaa acagaatttg 5520

acatgagatg ggtagcttaa tggctatgct gccacttctc gttattatta agttgaacta 5580

gcccgaagca aatcaactgg tgtttgtaag gttccttcca gcaccgaaac tatatacgcg 5640

gactaaggct ggctgcctga ggttaggccg gatgttgcct tcctttttgg gggaaaatcc 5700

caactggagg tgggattcaa ccggcttctt cccctcgttg acagcctcgc cttcacttct 5760

cactccacag ttgctagcat tcactctttc ggccttgctc cccactttgc tcttacgatc 5820

attttgccta cacgcagctg tgttgtctct gttgtcgtcc atccgcgtct gctctgatat 5880

tcttacccca acgcatcgtc aaaccaccac tgtcctaatc atggcttcct acaaagacgg 5940

aagccgtcaa gtacctctgc tccaggcttc tccagagg 5978

<210>15

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

ttcgacagcg tctccgacct 20

<210>16

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>16

acacagccat cggtccagac 20

<210>17

<211>5388

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>17

taaaatccgc cgcctccacc atttgtagaa aaatgtgacg aactcgtgag ctctgtacag 60

tgaccggtga ctctttctgg catgcggaga gacggacgga cgcagagaga agggctgagt 120

aataagccac tggccagaca gctctggcgg ctctgaggtg cagtggatga ttattaatcc 180

gggaccggcc gcccctccgc cccgaagtgg aaaggctggt gtgcccctcg ttgaccaaga 240

atctattgca tcatcggaga atatggagct tcatcgaatc accggcagta agcgaaggag 300

aatgtgaagc caggggtgta tagccgtcgg cgaaatagca tgccattaac ctaggtacag 360

aagtccaatt gcttccgatc tggtaaaaga ttcacgagat agtaccttct ccgaagtagg 420

tagagcgagt acccggcgcg taagctccct aattggccca tccggcatct gtagggcgtc 480

caaatatcgt gcctctcctg ctttgcccgg tgtatgaaac cggaaaggcc gctcaggagc 540

tggccagcgg cgcagaccgg gaacacaagc tggcagtcga cccatccggt gctctgcact 600

cgacctgctg aggtccctca gtccctggta ggcagctttg ccccgtctgt ccgcccggtg 660

tgtcggcggg gttgacaagg tcgttgcgtc agtccaacat ttgttgccat attttcctgc 720

tctccccacc agctgctctt ttcttttctc tttcttttcc catcttcagt atattcatct 780

tcccatccaa gaacctttat ttcccctaag taagtacttt gctacatcca tactccatcc 840

ttcccatccc ttattccttt gaacctttca gttcgagctt tcccacttca tcgcagcttg 900

actaacagct accccgcttg agcagacatc accatgcctg aactcaccgc gacgtctgtc 960

gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc 1020

gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat 1080

agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg 1140

ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc 1200

tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt 1260

ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc 1320

gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata 1380

tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt 1440

gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc 1500

cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata 1560

acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac 1620

atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg 1680

aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt 1740

gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt 1800

cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc 1860

agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga 1920

cgccccagca ctcgtccgag ggcaaaggaa tagagtagat gccgaccgcg ggatccactt 1980

aacgttactg aaatcatcaa acagcttgac gaatctggat ataagatcgt tggtgtcgat 2040

gtcagctccg gagttgagac aaatggtgtt caggatctcg ataagatacg ttcatttgtc 2100

caagcagcaa agagtgcctt ctagtgattt aatagctcca tgtcaacaag aataaaacgc 2160

gttttcgggt ttacctcttc cagatacagc tcatctgcaa tgcattaatg cattgactgc 2220

aacctagtaa cgccttncag gctccggcga agagaagaat agcttagcag agctattttc 2280

attttcggga gacgagatca agcagatcaa cggtcgtcaa gagacctacg agactgagga 2340

atccgctctt ggctccacgc gactatatat ttgtctctaa ttgtactttg acatgctcct 2400

cttctttact ctgatagctt gactatgaaa attccgtcac cagcncctgg gttcgcaaag 2460

ataattgcat gtttcttcct tgaactctca agcctacagg acacacattc atcgtaggta 2520

taaacctcga aatcanttcc tactaagatg gtatacaata gtaaccatgc atggttgcct 2580

agtgaatgct ccgtaacacc caatacgccg gccgaaactt ttttacaact ctcctatgag 2640

tcgtttaccc agaatgcaca ggtacacttg tttagaggta atccttcttt ctagaagtcc 2700

tcgtgtactg tgtaagcgcc cactccacat ctccactcga cctgcaggca tgcaagcttg 2760

gcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat 2820

cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat 2880

cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc 2940

cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct 3000

gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg 3060

gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 3120

tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc 3180

ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt 3240

cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat 3300

ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg 3360

agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt 3420

tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga 3480

gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa 3540

gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt 3600

attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt 3660

gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc 3720

agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga 3780

ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat 3840

cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct 3900

gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc 3960

cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 4020

gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 4080

ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 4140

acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca 4200

ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta 4260

aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc 4320

aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa 4380

ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca 4440

ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta 4500

actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc 4560

caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca 4620

gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta 4680

ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag 4740

cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt 4800

cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc 4860

acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac 4920

ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac 4980

gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc 5040

tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat 5100

accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag 5160

cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac 5220

gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc 5280

actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt 5340

gtgagcggat aacaatttca cacaggaaac agctatgacc atgattac 5388

Claims (10)

1. An Aspergillus niger genetically engineered bacterium is characterized in that a glucan synthesis regulation gene VosA in the strain is inactivated.

2. The genetically engineered bacterium of aspergillus niger according to claim 1, wherein the original aspergillus niger is aspergillus niger ATCC 12846.

3. The Aspergillus niger genetically engineered bacterium of claim 1, wherein the inactivated nucleotide sequence of the glucan synthesis regulatory gene VosA is shown in SEQ ID NO. 2.

4. The method for constructing the aspergillus niger genetically engineered bacterium of any one of claims 1 to 3, which is characterized by comprising the following steps:

(1) extracting the original genomic DNA of the Aspergillus niger;

(2) amplifying to obtain an upstream homology arm and a downstream homology arm of the gene VosA by using the genome DNA obtained in the step (1) as a template; amplifying to obtain a hygromycin resistance gene by taking a plasmid PAN7-1 as a template; taking an upstream homology arm and a downstream homology arm of the gene VosA and a hygromycin resistance gene as templates, and performing overlap extension PCR amplification to obtain a gene knockout fragment;

(3) and (3) introducing the gene knockout fragment obtained in the step (2) into an Aspergillus niger protoplast for homologous recombination to obtain the Aspergillus niger genetically engineered bacterium with the inactivated glucan synthesis regulatory gene VosA.

5. The use of the genetically engineered Aspergillus niger strain of any of claims 1-3 in the fermentative production of citric acid.

6. The application of claim 5, wherein citric acid is prepared by immobilized fermentation with the Aspergillus niger genetically engineered bacterium as a fermentation strain.

7. The use according to claim 6, wherein the immobilized fermentation uses a porous fibrous material as the immobilization medium.

8. The use according to claim 6, wherein the fermentation medium for immobilized fermentation is prepared by the following steps: weighing corn flour, adding water to prepare corn flour liquid with the concentration of 200g/L-300 g/L; adding 0.5mL-1mL of liquefying enzyme into every 1L of fermentation liquor, and preserving the heat at 60-75 ℃ for 35min-45 min; heating the corn powder liquid to 85-105 ℃, adding 0.5-1 mL of liquefying enzyme into every 1L of fermentation liquid, performing enzymolysis for 45-60 min until the iodine solution is not blue, and filtering the corn powder liquid to obtain a corn clear liquid; adding unfiltered corn flour liquid into the corn clear liquid, wherein the volume ratio of the unfiltered corn flour liquid to the corn clear liquid is 2-10:100, and uniformly mixing to obtain the immobilized fermentation medium.

9. The use according to claim 6, wherein the immobilized fermentation is under the following fermentation conditions: the fermentation temperature is 30-37 ℃, the fermentation time is 72-115 h, and the fermentation rotating speed is 200-330 rpm.

10. Use according to claim 7, wherein the porous fibrous material is a modified porous fibrous material, the modification steps being as follows: soaking the porous fiber material in absolute ethyl alcohol for 1-1.5 h, washing with water, and drying to constant weight to obtain the modified porous fiber material.

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