CN113817616B - Aspergillus niger genetically engineered bacterium with chitin synthase transcription regulation gene MedA inactivated and application thereof - Google Patents

Abstract

The invention discloses an Aspergillus niger genetically engineered bacterium inactivated by chitin synthase transcription regulatory gene MedA and application thereof. The invention constructs an Aspergillus niger genetically engineered bacterium with a chitin synthase transcription regulation gene MedA deletion through a gene knockout means. The genetically engineered bacterium reduces the amount of biomembrane of Aspergillus niger in the process of producing citric acid by immobilized fermentation, effectively relieves the phenomenon that the pore diameter of a carrier is blocked, and increases the oxygen and mass transfer effect of the carrier and the outside, thereby exerting the advantages of immobilized fermentation, improving the yield of citric acid, shortening the fermentation period, and effectively solving the problems of low yield and long fermentation period in the prior industrial Aspergillus niger fermentation production of citric acid.

Description

Aspergillus niger genetically engineered bacterium with chitin synthase transcription regulation gene MedA inactivated and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and in particular relates to an aspergillus niger genetically engineered bacterium inactivated by chitin synthase transcription regulation gene MedA, a construction method and application thereof.

Background

Citric acid is used as the first large acid in biochemical products, is widely applied to industries such as food, medicine, daily chemicals and the like, and is one of the organic acids with the largest worldwide demand and the most extensive application at present. China is the main production country of citric acid, and annual yield accounts for 53% of the total world yield. At present, the strain for producing citric acid in China is mainly filamentous fungi Aspergillus niger, and the substrate is mainly grain crude products such as tapioca flour, corn flour and the like. In recent years, the labor cost is generally increased due to the price of grains in China, and the production cost of citric acid is greatly increased. However, the rising cost makes the domestic citric acid market increasingly difficult due to the competitive citric acid products on the international market.

For decades, the industrial production of citric acid has mainly been in a liquid submerged fermentation mode. However, the liquid submerged fermentation has natural defects of poor tolerance, easy inactivation, easy aging, short catalytic aging and the like, can gradually decline the cell proliferation and differentiation capability of the aspergillus niger, has serious autolysis phenomenon, and cannot be continuously used for a long time, thereby causing accumulation of a large amount of thallus residues in the fermentation process, and causing the problems of nutrient waste and low substrate conversion rate. This is also one of the major bottlenecks in the current production technology in the citric acid industry. The cell immobilization fermentation technology based on the biological film can lead the fermentation to be continuously carried out, shortens the production period, improves the yield, greatly improves the efficiency of citric acid fermentation and reduces the cost. Under the environment, the cells can be gathered in a large scale with high density, and the characteristics of self-proliferation and self-repair intelligent characteristics, ecologically efficient mass and heat transfer system and long-term continuous reaction process are provided. The concept and the method overcome the defects of the prior method for fixing cells on a carrier by adopting the methods of polyvinyl alcohol gel embedding, glutaraldehyde crosslinking and the like, and also overcome the problems of serious autolysis phenomenon and limited thallus density improvement of deep liquid fermentation cells in a liquid phase. For the filamentous fungi, the adsorption method is simple to operate, has only physical effect in the fixing process, has no chemical change, can be theoretically used for fixing cells of all the filamentous fungi, and is beneficial to industrialized large-scale application.

Disclosure of Invention

The invention aims to: the invention aims to solve the technical problem of providing an aspergillus niger genetically engineered bacterium aiming at the defects of the prior art.

The invention also solves the technical problem of providing a construction method of the Aspergillus niger genetically engineered bacterium.

The invention further aims to provide the application of the aspergillus niger genetically engineered bacterium in producing citric acid.

In order to solve the first technical problem, the invention discloses an aspergillus niger genetically engineered bacterium, wherein a chitin synthase transcription regulatory gene MedA in the strain is inactivated.

The Aspergillus niger genetically engineered bacterium replaces the genetically engineered bacterium with a hyg resistance gene (hygromycin resistance gene) to replace part of the sequence of the gene MedA by a double-exchange method, and the transcription regulating gene MedA can activate the expression of the chitin synthase A, B, C gene in Aspergillus niger to generate chitin which is secreted outside cells and is an important component of extracellular matrixes. Knocking out the MedA gene can lead to the fact that the chitin synthase A, B, C gene cannot be activated and the synthesis of chitin is inhibited, so that the formation of extracellular matrixes of the Aspergillus niger is reduced, excessive biological films can not be formed in the immobilization process of the Aspergillus niger to influence oxygen transfer and mass transfer, and the immobilization fermentation effect is remarkably improved.

Wherein the original Aspergillus niger is Aspergillus Niger ATCC12846.

Wherein, the nucleotide sequence of the chitin synthase transcription regulating gene MedA inactivation is shown as SEQ ID NO. 2, and the nucleotide sequence of the chitin synthase transcription regulating gene MedA that is not inactivated is shown as SEQ ID NO. 1.

In order to solve the second technical problem, the invention also discloses a preparation method of the Aspergillus niger genetically engineered bacterium, which comprises the following steps:

(1) Extracting genomic DNA of original aspergillus niger ATCC12846;

(2) Amplifying the genome DNA obtained in the step (1) to obtain an upstream homology arm and a downstream homology arm of the gene MedA; the plasmid PAN7-1 is used as a template to amplify and obtain hygromycin resistance genes; the gene knockout fragment is obtained by overlapping extension PCR amplification by taking an upstream homology arm, a downstream homology arm and a hygromycin resistance gene of the gene MedA as templates;

(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 chitin synthase transcription regulation gene MedA inactivated.

In the step (2), the nucleotide sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4 are used as primers when amplifying homologous arms on the upstream of the gene MedA; when the downstream homology arm of the gene MedA is amplified, the nucleotide sequences shown in SEQ ID NO. 5 and SEQ ID NO. 6 are used as primers; 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 gene knockout fragment is amplified by overlap extension PCR, the nucleotide sequences shown in SEQ ID NO. 9 and SEQ ID NO. 10 are used as primers.

In the step (2), the nucleotide sequence of the upstream homology arm of the gene MedA is shown as SEQ ID NO. 11, the nucleotide sequence of the downstream homology arm of the gene MedA 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.

In order to solve the third technical problem, the invention also discloses application of the Aspergillus niger genetically engineered bacterium in producing citric acid by fermentation, wherein the Aspergillus niger genetically engineered bacterium is used as a fermentation strain, and the citric acid is prepared by immobilized fermentation.

Wherein the immobilized fermentation takes porous fiber material as an immobilized carrier, and polyurethane is preferable.

Wherein the immobilized carrier is pretreated by alkali soaking and acid soaking in sequence, preferably, the immobilized carrier is soaked in 0.6-1.8M NaOH for 45-90min, washed with water until the pH is neutral, soaked in 0.6-1.8M HCl for 45-90min, washed with water until the pH is neutral, and dried to obtain the immobilized carrier.

Wherein the immobilized carrier is sheared into the same size and the volume is 0.5cm ³ Is a square block.

Wherein the dosage of the immobilized carrier in the immobilized fermentation is 0.5-3g/L fermentation medium, preferably 2g/L fermentation medium.

Wherein, in the fermentation, the preparation method of the fermentation medium comprises the following steps: incubating the aqueous corn flour solution with a liquefying enzyme at 63-72deg.C for 38-43min; heating to 88-102 deg.C, and performing enzymolysis with liquefying enzyme until iodine solution does not change blue to obtain corn flour liquefied solution; filtering to obtain clear corn liquid; mixing the obtained corn clear liquid with the corn flour liquefaction liquid uniformly to obtain the corn flour liquefaction liquid; preferably, 220-280g/L corn flour aqueous solution is subjected to liquefying enzyme (0.5-1.5 mL/L corn flour aqueous solution) and is kept at 63-72 ℃ for 38-43min; heating to 88-102 deg.C, and performing enzymolysis with liquefying enzyme (0.6-1.5 mL/L corn flour water solution) until iodine solution is not blue to obtain corn flour liquefied solution; filtering to obtain clear corn liquid; mixing the obtained corn clear liquid with the corn flour liquefaction liquid according to the volume ratio of 100 (4-8).

Wherein the liquefying enzyme contains alpha-amylase, the enzyme activity of the alpha-amylase is 72000-78000U/mL (1 mL of enzyme liquid is 1 enzyme activity unit, U/mL, which is required for hydrolyzing 1mg of soluble starch for 1min under the conditions of pH5.5 and temperature of 85 ℃).

Wherein, the fermentation is to scrape the activated Aspergillus niger genetic engineering bacteria flat plate with a spore scraping liquid to prepare spore liquid, inoculate the spore liquid in a fermentation culture medium containing an immobilized carrier, and ferment to obtain the citric acid.

Wherein, the spore liquid is inoculated according to the volume ratio of 0.1% -10%, preferably 0.1% -5%, and more preferably 0.5%.

Wherein, the conditions of the fermentation are as follows: the fermentation temperature is 32-37 ℃, the fermentation time is 74-117h, and the fermentation rotating speed is 220-350rpm.

The beneficial effects are that: compared with the prior art, the invention has the following advantages:

the invention constructs an Aspergillus niger genetically engineered bacterium with a chitin synthase transcription regulation gene MedA deletion through a gene knockout means. The genetically engineered bacterium reduces the amount of biomembrane of Aspergillus niger in the process of producing citric acid by immobilized fermentation, effectively relieves the phenomenon that the pore diameter of a carrier is blocked, and increases the oxygen and mass transfer effect of the carrier and the outside, thereby exerting the advantages of immobilized fermentation, improving the yield of citric acid, shortening the fermentation period, and effectively solving the problems of low yield and long fermentation period in the prior industrial Aspergillus niger fermentation production of citric acid.

Drawings

The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.

FIG. 1 is an electrophoretogram of the A.niger Aspergillus Niger ATCC12846 genome, where M is DNAMaroker at DL10000 and lane 1 is the A.niger genome.

FIG. 2 is a PAN7-1 plasmid map.

FIG. 3 shows PCR electrophoresis of upstream and downstream homology arms of the MedA gene, wherein M is DNAMmarker of DL5000, lane 1 is upper homology arm of MedA, and lane 2 is lower homology arm of MedA.

FIG. 4 is a hyg resistance expression element, wherein M is DNAMaroer of DL5000, lane 1 is a hyg resistance expression element.

FIG. 5 is an electrophoretogram of a gene knockout fragment, wherein M is DNAMaroker of DL10000, and lane 1 is a gene knockout fragment.

FIG. 6 is a crystal violet staining chart.

FIG. 7 is a graph showing the difference in the OD values of crystal violet staining.

FIG. 8 shows fermentation results of original Aspergillus niger and Aspergillus niger genetically engineered bacteria.

FIG. 9 shows the results of the difference in production intensity after 8 batches of original Aspergillus niger and Aspergillus niger genetically engineered bacteria were fermented continuously.

FIG. 10 is a diagram of the vector after 96h of continuous fermentation of original Aspergillus niger and Aspergillus niger genetically engineered bacteria.

Detailed Description

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.

Example 1 construction of Aspergillus niger iron acquisition regulatory Gene MedA knockout bacterium

(one) extraction of the original Aspergillus niger genome

A kit (takara minibest plant genomicDNA extraction kit) for extracting plant genome from takara corporation was used, and the specific method was as follows:

1. 1mL of scraped Aspergillus niger ATCC12846 spore liquid is inoculated into 50mL of DP culture medium, and the culture is carried out for 24 hours at 35 ℃ and 200 r/min; the formula of the DP medium 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 mixture is subpackaged into 50mL after water is used for sizing to 100mL.

2. Centrifuging the product obtained in the step 1 at 8000r/min for 5min, collecting mycelium pellets, washing twice with normal saline, grinding the collected mycelium pellets with liquid nitrogen for 3 times, weighing 100mg of ground powder, adding into a centrifuge tube with 500 mu L of Buffer HS II added in advance, uniformly mixing, adding 10 mu L of RNase A, fully vibrating and uniformly mixing, and carrying out water bath at 56 ℃ for 10min.

3. And (2) adding 62.5 mu L of Buffer KAC into the product obtained in the step (2), and fully and uniformly mixing. Place on ice for 5min and centrifuge at 12000rpm for 5min. 600. Mu.L of the supernatant was taken, 600. Mu.L of Buffer GB was added thereto, and the mixture was thoroughly mixed.

4. The separation column was placed in a collection tube, the solution obtained in step 3 was transferred to the separation column, centrifuged at 12000rpm for 1min, and the filtrate was discarded.

5. 500. Mu.L of Buffer WA WAs added to the separation column in step 4, and the mixture WAs centrifuged at 12000rpm for 1min, and the filtrate WAs discarded.

6. 700. Mu.L of Buffer WB was added to the column in step 5, centrifuged at 12000rpm for 1min, and the filtrate was discarded.

7. The step 6 is repeated once.

8. The separation column in step 7 was mounted on a collection tube and centrifuged at 12000rpm for 2min.

9. The separation column in step 8 was placed on a new 1.5mL centrifuge tube, 40. Mu.L of 65℃sterilized water was added to the center of the separation column membrane, and the mixture was allowed to stand at room temperature for 1min. The DNA was eluted by centrifugation at 12000rpm for 2min. The genomic concentration of A.niger as determined by agarose gel electrophoresis is shown in FIG. 1. FIG. 1 shows that M is a DNA marker of DL10000 and that No. 1 is an extracted genome of Aspergillus niger.

(II) PCR technique is used to amplify homologous arm of gene MedA upstream and downstream

Amplifying an upstream homology arm by taking the original Aspergillus niger genome extracted in the step (I) as a template, taking MedA-up-F as an upper primer and taking MedA-up-R as a lower primer (the nucleotide sequence of MedA-up-F is shown as SEQ ID NO:3 and the nucleotide sequence of MedA-up-R is shown as SEQ ID NO: 4); the downstream homology arm is amplified by using MedA-Down-F as an upper primer and MedA-Down-R as a lower primer (the nucleotide sequence of MedA-Down-F is shown as SEQ ID NO:5, and the nucleotide sequence of MedA-Down-R is shown as SEQ ID NO: 6). The reaction system is shown in Table 1, and the PCR reaction conditions are as follows: (1) denaturation at 95℃for 10s; (2) annealing at 55 ℃ for 30s; (3) Extending at 72 ℃ for 5min, and repeating the steps (1) to (3) 35 times.

TABLE 1 PCR reaction System and reaction conditions

After the reaction, the PCR products were quantified by agarose gel electrophoresis, see FIG. 3, M was the DNA Marker of DL5000, the upper homology arm No. 1 was the MedA, and the lower homology arm No. 2 was the MedA. The nucleotide sequence of the upstream homology arm of the gene MedA is shown as SEQ ID NO. 11, and the nucleotide sequence of the downstream homology arm of the gene MedA is shown as SEQ ID NO. 12.

(III) amplification of hyg-resistant expression elements

The hyg resistant expression element was amplified using a PAN7-1 plasmid (stored in the laboratory, the PAN7-1 plasmid map is shown in FIG. 2, the nucleotide sequence is shown in SEQ ID NO: 17), medA-hyg-F as the upper primer, and MedA-hyg-R as the lower primer (the MedA-hyg-F nucleotide sequence is shown in SEQ ID NO:7, and the MedA-hyg-R nucleotide sequence 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 95℃for 10s; (2) annealing at 55 ℃ for 30s; (3) Extending at 72 ℃ for 5min, and repeating the steps (1) to (3) 35 times. After the reaction, the PCR products were quantified by agarose gel electrophoresis, see FIG. 4, M was DNA Marker of DL5000, and hyg resistance expression element No. 1. The nucleotide sequence of the hyg resistance expression element is shown as SEQ ID NO. 13.

(IV) amplified Gene knockout fragment

The up-stream homology arm, the down-stream homology arm and hyg resistance expression element of the MedA are used as templates, the MedA-F is used as an upper primer, the MedA-R is used as a lower primer (the nucleotide sequence of the MedA-F is shown as SEQ ID NO. 9, the nucleotide sequence of the MedA-R is shown as SEQ ID NO. 10), and the overlapping extension PCR (Overlap PCR) technology is used for amplifying the MedA gene knockout fragment. The reaction system is shown in Table 1, and the PCR reaction conditions are as follows: (1) denaturation at 95℃for 10s; (2) annealing at 55 ℃ for 30s; (3) Extending at 72 ℃ for 5min, and repeating the steps (1) to (3) 35 times. After the reaction, the PCR products were quantified by agarose gel electrophoresis, see FIG. 5, M was DNA Marker of DL10000, and number 1 was a gene knockout fragment. The nucleotide sequence of the gene knockout fragment is shown as SEQ ID NO. 14.

Preparation and transformation of Aspergillus niger protoplast

1. Aspergillus niger ATCC12846 was inoculated onto PDA plates, spores were grown, 3mL of a curettage buffer was added to the plates, spores were scraped off with a spreader bar, and transferred to a sterilized 5mL centrifuge tube.

The culture medium formula of the PDA plate is as follows: weighing 200g peeled potato, and cutting into 1cm ³ Adding 600mL of water into small blocks with the size, boiling for 30min, filtering with 4 layers of gauze, metering the volume of the obtained potato juice to 1L with a measuring cylinder, and packaging. Then 20g/L glucose and 15g/L agar powder are added, and the mixture is sterilized for 20min at 115 ℃.

2. Inoculating 0.5mL of spore liquid to 50mL of DP culture medium, culturing at 35 ℃ and 200rpm for 13-16h, and observing the spore germination state by microscopic examination.

3. After the spores germinate, they were filtered through a Miracloth filter cloth (Miracloth), leaving the mycelia behind.

An enzymatic hydrolysate (lyase (Sigma L1412), a crashing enzyme Driselase (Michelin D861435), snailase (Shanghai Biotechnology A600870) was prepared at 0.1g/10mL each, and cellulase (Sigma C2605) at 400. Mu.L/10 mL) and sterilized by filtration through a sterile syringe.

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

5. After the enzymolysis is completed, filtering with filter paper, taking filtrate, and centrifuging at 5000rpm for 10min at 4 ℃. The supernatant was removed, 1mL of a 1M aqueous sorbitol solution (ice water bath) was added, the mixture was blown and sucked with a gun, 15mL of a 1M aqueous sorbitol solution was added, and the mixture was centrifuged to remove the supernatant. And then repeated one more time. The supernatant was removed, 1mL of solution 5 was added, and the mixture was blown and sucked with a gun to obtain protoplasts. Wherein the solution 5 is self-made and comprises the following formula: KCl 4.47g, caCl ₂ 0.735g,MOP 0.2093g pH was adjusted to 6.0 with KOH and volume was set to 100mL with water.

6. 100. Mu.L of protoplasts were pipetted into a 1.5mL sterile centrifuge tube,adding 10 mu L of the gene knockout fragment constructed in the step (four) and mixing the mixture. Then 50. Mu.L of solution 4 is added, mixed well, placed on ice and timed for 15-30min. Wherein the solution 4 is prepared by self and comprises the following formula: PEG8000 25g, caCl ₂ 1.47g,KCl 4.47g,10mM Tris pH is adjusted to 7.5 with hydrochloric acid and volume is adjusted to 100mL with water.

After 7.20min, 900. Mu.L of solution 4 was added, mixed up and down several times, left at room temperature and timed for 15-30min. After 15-30min, centrifuge at 6000rpm for 5min, discard 900. Mu.L supernatant, spread the rest thallus on PDA culture medium with sucrose concentration of 1mol/L and hygromycin concentration of 150mmol/L, and forward culture to obtain transformant.

8. Transformants were picked for colony PCR validation. The specific method comprises the following steps: a proper amount of the transformant was added to 50. Mu.L of colony PCR buffer (100 mM/L Tris-HCl, 10mM/L EDTA, 1M/L KCl), and the mixture was subjected to a 95℃water bath for 10 minutes to give 0.5. Mu.L of the transformant, which was then added to the PCR reaction system. The PCR primers are hyg-F and hyg-R (the nucleotide sequence of hyg-F is shown as SEQ ID NO:15, the nucleotide sequence of hyg-R is shown as SEQ ID NO: 16), and agarose electrophoresis shows amplified bands, which indicate successful transformation, thus obtaining the delta MedA strain.

Example 2 Crystal Violet staining experiments

Inoculating original Aspergillus niger (ATCC 12846) and genetically engineered bacterium (delta MedA strain) to a PDA plate respectively, adding 3mL of spore scraping buffer solution to the plate after spores grow, scraping the spores by a coating rod, transferring to a sterilized 5mL centrifuge tube, and fixing the volume to 2mL by the spore scraping buffer solution to obtain spore liquid. Quantitated by a hemocytometer and diluted to 10 ⁴ individual/mL, followed by continued dilution to 10 ³ /mL，10 ² /mL。

1mL of synthetic medium was previously added to a 24-well plate, and then 2. Mu.L of spore solutions of different concentrations were inoculated into the medium. And (3) standing and culturing at 35 ℃ for 72 hours to enable the aspergillus niger to form a film at the bottom of the pore plate. After that, the medium was poured off, washed 2 times with PBS, and stained with 0.1% crystal violet for 15min. And pouring out crystal violet, washing with PBS for 2 times, adding glacial acetic acid, and placing in a shaker for 30min to decolorize the crystal violet. Then observation is performed and the microplate reader detects the OD570. The crystal violet staining pattern and the OD value difference pattern are shown in fig. 6 and 7.

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

TABLE 2 OD values of biofilm Crystal Violet staining experiments at different spore concentrations

The results in FIGS. 6 and 7 show that the ΔMedA strain, after decolorization, is significantly lighter in purple than the original strain, at 10 ² The color of the delta MedA strain at the concentration is removed, the OD value detected by the enzyme label instrument is consistent with the color. Indicating that the biofilm is reduced after the inactivation of the chitin synthase regulatory gene Δmeda.

Example 3 immobilized fermentation experiment of genetically engineered bacteria

1. Preparation of porous fibrous material immobilization medium

The polyurethane material (molecular weight 3000-3500) is soaked in NaOH of 1M for 1h, washed with pure water, soaked in 1MHCl for 1h, then washed with pure water until the pH is neutral, and then put into an oven of 65 ℃ for drying to constant weight. Cut into 0.5cm ³ Vectors of the same size on the left and right.

2. Preparation of fermentation Medium

Weighing 250g of corn flour, adding 1L of water, uniformly mixing to prepare a corn flour aqueous solution, placing in a water bath kettle at 75 ℃, adding 1mL of liquefying enzyme (60000U/L) when the corn flour aqueous solution reaches 65 ℃, liquefying for 40min, heating the water bath kettle to 95 ℃, adding 1mL of liquefying enzyme when the corn flour aqueous solution reaches 85 ℃, liquefying for 60min until iodine solution does not turn blue, and obtaining corn flour liquefied solution; filtering the corn flour liquid to obtain corn flour clear liquid; adding unfiltered corn flour liquefied liquid into corn flour clear liquid as return material (the volume ratio of unfiltered corn flour liquid to corn clear liquid is 8 percent), mixing uniformly, and taking the mixture as fermentation medium ]. 100mL of the well-mixed fermentation medium was dispensed into 500mL Erlenmeyer flasks containing 0.2g of the carrier. Sterilizing and cooling for standby.

3. Immobilized continuous fermentation

(1) Inoculating frozen Aspergillus niger genetically engineered bacteria and original Aspergillus niger spores to PDA plate, culturing at 35 deg.C for 4-5 days, and culturing until spores grow on the PDA plate.

(2) The spores were scraped with a spore scraping buffer to obtain a spore suspension, a proper amount of the spore suspension was transferred to a 500mL conical flask containing 100mL immobilized medium, cultured for 96 hours at 35℃in a shaking table at 250rpm, sampled every 12 hours during fermentation, centrifuged at 12000rpm for 5 minutes, the supernatant was separated from the pellet, and the residual sugar concentration and citric acid yield in the supernatant were measured. When the residual sugar concentration is lower than 5g/L, the first batch of immobilized fermentation is finished. Wherein, the citric acid is measured by NaOH titration and the total sugar is measured by DNS method.

(3) After the first batch fermentation is finished, pouring out the fermentation liquor, keeping the carrier in a bottle, pouring a new sterilized culture medium into a conical flask, performing immobilized continuous fermentation of a second batch, and repeating the fermentation for 8 batches under the same culture conditions as in the step (2).

Wherein, the NaOH titration method is as follows: 1mL (diluted to a certain concentration) of the sample was taken and added to a 250mL Erlenmeyer flask, and 50mL of pure water was added at the same time, and titrated with 0.1429M NaOH, the amount of NaOH consumed being the yield of citric acid.

Wherein, the DNS method is as follows:

preparing DNS: 10g of 3, 5-dinitrosalicylic acid is weighed and placed in about 600mL of water, 10g of sodium hydroxide is gradually added, the solution is magnetically stirred and dissolved in a water bath at 50 ℃,200 g of sodium methyl tartrate, 2g of phenol and 5g of anhydrous sodium sulfite are sequentially added, after all the solution is dissolved and clarified, the solution is cooled to room temperature, and pure water is used for fixing the volume to 1000mL. Stored in brown reagent bottles and used after 7d of storage in the dark.

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 taking 0.5mL of each sugar standard solution into 15mL centrifuge tubes, then adding 0.5mL of DNS solution into each centrifuge tube,

the centrifuge tubes were placed in a boiling water bath for reaction for 5min, then placed in ice water for cooling, then 8mL of pure water was added to each centrifuge tube, and after mixing, the absorbance OD540 at a wavelength of 540nm was measured, and 0.0g/L was used as a control. The concentration of the standard solution is on the ordinate, and the absorbance OD540 is on the abscissa, and a standard curve is drawn.

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

High performance liquid chromatography to determine citric acid:

and (3) manufacturing a standard curve: and (3) measuring the content of the citric acid by adopting high performance liquid chromatography, preparing a series of citric acid standard solutions with the concentration of 0-10g/L, sucking 1mL of a filter membrane with the concentration of 0.22 mu m, filtering, and filling the filter membrane into a liquid phase vial for measurement. The analysis conditions were as follows: a RID differential detector; the mobile phase was 5mM H2SO4; the flow rate of the Aminex HPX-87H organic acid column of Bio-Rad is 0.6mL/min; the sample injection amount is 20 mu L, the column temperature is 55 ℃, and the stop time is 20min. The peak areas of samples of different concentrations were determined. And drawing a standard curve by taking the concentration of the standard solution as an ordinate and the corresponding peak area as an abscissa.

The measuring method of the sample comprises the following steps: the supernatant was diluted appropriately, sucked into 1ml of a 0.22 μm filter membrane, and then put into a liquid phase vial for measurement. The measured peak areas were taken into the standard curve to calculate the concentration.

FIG. 8 shows the difference between the ΔMedA strain and the original strain under fermentation conditions, and shows that the yield of citric acid produced by fermentation of the ΔMedA strain after 96 hours of fermentation is 181.6g/L on average, and the yield of citric acid produced by fermentation of the original strain is 161.4g/L on average. In the conversion rate, the conversion rate of the delta MedA strain reaches 100.9 percent, and the conversion rate is improved by 12.5 percent compared with the original strain. FIG. 9 shows the difference in production intensity between the ΔMedA strain and the original strain after 8 batches of continuous fermentation. Meanwhile, the period of the knocking out bacteria is reduced from 96 hours to 60 hours, and is reduced by 37.5 percent. The fermentation strength is improved from 1.68g/L/h to 3.03g/L/h, and the fermentation strength is improved by 80.3 percent. As can be seen from the graph, the ΔMedA strain has no obvious decrease in fermentation strength after 8 batches of continuous fermentation, while the original strain has a 30.5% decrease in fermentation strength after 8 batches, indicating that the ΔMedA strain has significantly better continuous fermentation performance than the original strain. Meanwhile, as can be seen in Table 3, the total content of the hetero acids in the immobilized fermentation broth of the DeltaMedA strain is reduced from 4.8g/L to 2.9g/L of the original strain by 39.6%. Compared with other genetically engineered bacteria, the content of the mixed acid is obviously reduced by 19.4 percent. After the MedA gene is knocked out, the amount of bacteria adsorbed on the carrier is further reduced, so that the oxygen transfer and mass transfer effects in the pore diameter of the carrier are obviously enhanced, by-products such as oxalic acid, malic acid and the like generated under the severe fermentation environment and in an anoxic state are reduced, and the quality of a citric acid finished product is obviously improved. Meanwhile, compared with the original strain, the yield, the conversion rate and the production strength of the delta MedA strain are remarkably improved, multi-batch continuous fermentation can be realized, the production strength is not obviously reduced, so to say that great progress is made on the basis of immobilized fermentation of Aspergillus niger into citric acid production, and the method is suitable for industrial production.

TABLE 3 hetero acids in fermentation broths

Wherein, the carrier after the immobilized fermentation for 4d is taken out, and the adsorbed hypha is washed off by washing 3 times with PBS. As shown in FIG. 10, the amount of the original bacteria was large, the pore diameter of the carrier was almost filled, and the amount of the knock-out bacteria was moderate.

The invention provides an Aspergillus niger genetically engineered bacterium inactivated by chitin synthase transcription regulation gene MedA, and an application thought and a method thereof, and particularly the method and the way for realizing the technical scheme are numerous, the above is only a preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made by a person of ordinary skill in the art without departing from the principle of the invention, and the improvements and the modifications are also regarded as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.

Sequence listing

<110> university of Nanjing Industrial science

COFCO Biotechnology Co.,Ltd.

<120> A strain of Aspergillus niger genetically engineered bacterium inactivated by chitin synthase transcription regulatory gene MedA and application thereof

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<170> SIPOSequenceListing 1.0

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<211> 2056

<212> DNA

<213> Aspergillus niger (Aspergillus niger)

<400> 1

cacggtgttt tggtgagtcc gggtcttcct gctcgctgcc caattgttgc tttttggtcc 60

cttcccattc cattttgttt ttcgactgga tcagaatatc tgattcatca ctgtctcgcc 120

gagtgcccca gccttgtttt tcctgcagtc acacccacct tctcacccgc cgaacactga 180

actgacccgg gggcgtttta cagttcaatt ccgagttaca aacaccgtta attctagacg 240

agtcgttgga tttttcggat cctgaagagt cacagtttct cggtgctttt tcagacggcc 300

ttctggccaa gtctgagccc atcttgtcta tgtctacttt ccagaagccc ccgcacgcgg 360

cgttgatcgg taagcactga tacaccccct cccccctccc ccttcctcgt gcacatggca 420

tggctgggcc aggggtccct agggaaaacg cactccgttt gaactctgtg agacgctgac 480

gctcaacttt acctgaatag actacgactc tgctcgatct ttgcatgatg gtattcctta 540

ccaaggctat ggccagacac cctatgtgac ggccacgccg ctggttcctt cccccatgcc 600

cgatcacgca agccagatat cggactgtgt gccttacatg gcgaacggag agtatgcgag 660

ctcctacgaa gaggctcgtt caccaatgct ggccgccgag aaccgccaga tgccggaagt 720

tgtctcttat tccccccaaa gaggatccga aggtaccaga gtcttcgtcc agattcaatc 780

gccctatgat ctccatacat cctcctatgc gactctttac cttgtcttcg gctcgaagaa 840

atgtgaatgc gtcccccact tcctcggctt ccagggtgct tcattccagt atgcgctttc 900

cgtcgacacg ccccctttca gctccacggg ctctccatcg ttcgcagttc ccttgcaagt 960

gtccatggat cacaacgact gccatgcagt cacgctacaa gtgggtgttt atacctatga 1020

acatgcctcg atgcagtcgc cttcagatga gtcccgcaag cggagggtgc cttcatacgc 1080

ggatgaacct ctgtcgaggg cacccaaacg aatcaccggc ccaccaatgc atgcaaagga 1140

accatccagc gtatcctcgg cgacctatcc gccgtacctg cagtctttgc ccgcagtgaa 1200

tagtttcgtc agcccacatc atacggcggc atcgccgaga gttccttcga cgcagtatcc 1260

cggcctatct accgcatctc aagcctctat ccgggcgccg tcgcctatga ctccatcata 1320

cagcccgtcc ttccttgctg tcagcaacga tgcccgaagc actacttata ccatcggcca 1380

tcctctgcgt cagcaaaacc aagcatcccc aagcaggttc gggaatccaa cgctcattcg 1440

aacgtccacg ttgcagcagt cgggcgcata tggccaaacg cagtcgttca acccctatgc 1500

catgtgcccg accaaggcag tgttgaagtt gagtggtgat cttgattcaa tgacggaaag 1560

ctggtccaga gaagagcggg aggcgaagcg tcggttggtc caattcacac ggatgcaaag 1620

cggcagtact atccacgcag atttcaagcc tgtggcgcct gaagatcgtg ctcccaatag 1680

catctgcatc agctgcatct actgggatgg gaaagacgaa tgctttgtta ccagtgaban 1740

dnsaccagcc ctattcttcc gccttggccg cgccttactc gtacccttct gtctgtcagc 1800

agcagagcca actcgggctc acagccaccg tgaacagcag aaattgggac ttgaatcctc 1860

ttctcaactc tgctgcagcc actggcaatc ccagcagcag cgcgtgctac aactaccttg 1920

ctcccatgcc gtattctgtg catggccagg cccacgggtc ttgaaatacc tgcgcgtttt 1980

ggttcctcca tggtaatctg catcgactgg cgtttggctt tcccgcttta ctgcgttgtg 2040

atttcttgca taattt 2056

<210> 2

<211> 3228

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

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 gagaagagcg ggaggcgaag 2760

cgtcggttgg tccaattcac acggatgcaa agcggcagta ctatccacgc agatttcaag 2820

cctgtggcgc ctgaagatcg tgctcccaat agcatctgca tcagctgcat ctactgggat 2880

gggaaagacg aatgctttgt taccagtgab andnsaccag ccctattctt ccgccttggc 2940

cgcgccttac tcgtaccctt ctgtctgtca gcagcagagc caactcgggc tcacagccac 3000

cgtgaacagc agaaattggg acttgaatcc tcttctcaac tctgctgcag ccactggcaa 3060

tcccagcagc agcgcgtgct acaactacct tgctcccatg ccgtattctg tgcatggcca 3120

ggcccacggg tcttgaaata cctgcgcgtt ttggttcctc catggtaatc tgcatcgact 3180

ggcgtttggc tttcccgctt tactgcgttg tgatttcttg cataattt 3228

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

acttccactc gctcgtccca 20

<210> 4

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

ggtggaggcg gcggatttta ttggccagaa ggccgtctga 40

<210> 5

<211> 42

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

cccactccac atctccactc gagagaagag cgggaggcga ag 42

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

agtaggcgac cagagcagca 20

<210> 7

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

tcagacggcc ttctggccaa taaaatccgc cgcctccacc 40

<210> 8

<211> 42

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

cttcgcctcc cgctcttctc tcgagtggag atgtggagtg gg 42

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

ctttacgcgc tcgctgattg 20

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

gtgggtttga gtcaccgagg 20

<210> 11

<211> 1883

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

ctttacgcgc tcgctgattg ttcgacaccc gtcgctatat tctctgttta attgcctcga 60

agcttggctc cgtctccatc cgaccagaag gggggaggat ctttttctta ttttattttc 120

gacaatagtg tctttaatat ttccgaagca accgaaaatc cacgggaaag ccgctgattg 180

actggacgcc atcttctttc cgtgtcgtgt ggtctcctac ccagctgttc gtttgcctgg 240

tcccttatac gccggatcta tagagcgatc tccgtttgtt gagcggaagg tacccccaag 300

gacgatcatc gaaccaccgt cttttgtcac gtccaacaac cctcctcgtt tcagcaaaat 360

cacctttatt tgcagtgtcc ggattcgctc atctgtctca attttacact ctcccgaaca 420

gaaaacgaga gtggatcgtc agctagccat cttgccaacc ttgtctctct gtagtccttc 480

acacgaggtc ggacctggac tcaaaccatc gtggatcttt gtctcggctg aggacaccac 540

cttattacga actagcaggg aaatatctca aataggctgc gtttactgac gaagcctctc 600

cctgtgctga agtcattggt ctgttaaggt tggtgccaac ccccccctcc ctcgcccttt 660

gccacagact ctgttggtgc cttgcatcac tctccttcgt gtccccttcc ctctgtactc 720

cttgcttctc caaggtcgtg cctttgccgc acaagatccc aaggaaaatt agggtccacg 780

atgggttccg tgcgcttgtc acctcccctt gtatggtttc cgtcactaaa cttccggatg 840

tttgttgtta acccgccccc gtcaaggtgt agactacctc gtttagtcgc ttagggaccg 900

tgaccttttt tgcatattag atttcaaaag acctcagaga cgcatctctt gtcttctcat 960

ctcacctggt aagtcttgct ggtctatgca gcgagccggt tcctcgcctt ttgggagggt 1020

tcttactcac tagttgagta gttcgtgccc cgacctagtc ctcatcatcc tgagggaaac 1080

ttgagacaat ttcgcttcca ggaacgccag aaaagcgaaa aggtcttctt ctcacttcga 1140

ccggctcttc tcgctcgctc ttatccagtg cctgttacgg atatctgcca tccaacctat 1200

ccgccgcgtg aggttaaaac agcgccttca gatggaagct ggtttcctat aggttgtcag 1260

aagcattcat cgacagtgtg gctcacctct ggtctctatc ctgcagccaa cctttcgtgg 1320

tttcaccctg aggcgttgga tcctgatcgc tgattcgaag cttttcgtgg catcttcctc 1380

gtccgggctg acagccactt cgtgtcgtca ttcgttttgt ctcggagagc tcgctagtaa 1440

ctttccgaca tcctatcgtt ttccaggata aattcgctgt cattcgcttt ttttaagctg 1500

ttgtgcttcg ttgcagtttc tgcaacacct tcaaactggc ttactcgaag acgcacggtg 1560

ttttggtgag tccgggtctt cctgctcgct gcccaattgt tgctttttgg tcccttccca 1620

ttccattttg tttttcgact ggatcagaat atctgattca tcactgtctc gccgagtgcc 1680

ccagccttgt ttttcctgca gtcacaccca ccttctcacc cgccgaacac tgaactgacc 1740

cgggggcgtt ttacagttca attccgagtt acaaacaccg ttaattctag acgagtcgtt 1800

ggatttttcg gatcctgaag agtcacagtt tctcggtgct ttttcagacg gccttctggc 1860

caataaaatc cgccgcctcc acc 1883

<210> 12

<211> 1463

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

cccactccac atctccactc gagagaagag cgggaggcga agcgtcggtt ggtccaattc 60

acacggatgc aaagcggcag tactatccac gcagatttca agcctgtggc gcctgaagat 120

cgtgctccca atagcatctg catcagctgc atctactggg atgggaaaga cgaatgcttt 180

gttaccagtg abandnsacc agccctattc ttccgccttg gccgcgcctt actcgtaccc 240

ttctgtctgt cagcagcaga gccaactcgg gctcacagcc accgtgaaca gcagaaattg 300

ggacttgaat cctcttctca actctgctgc agccactggc aatcccagca gcagcgcgtg 360

ctacaactac cttgctccca tgccgtattc tgtgcatggc caggcccacg ggtcttgaaa 420

tacctgcgcg ttttggttcc tccatggtaa tctgcatcga ctggcgtttg gctttcccgc 480

tttactgcgt tgtgatttct tgcataattt ttgatacctc tttgattatc gtcactcctt 540

aaccgcgaga ttttggcact gagccctgtg gattttctag cacaggataa ctcagtcacg 600

ggcctcacgg actttaccgt ttatttttca tgatcactgt gaatgatctc atgtctctcc 660

ttataccatc gcccttcttc atgccttaat gcccgatatc cttttttttc tttgacgcat 720

tatagacgaa ctgttgctgt tcttcaccat ttacaccatg tttttatttt ttgtggttaa 780

tgttgcattt tcatgtgggc atgaggcttt cgctccttta cgaagctgta caactatctg 840

gcatagcatg gcgctgccgt cgttccttct aggttgcagg tttgggataa cacttgggat 900

tgatgtacca gacatctggg ttggcgaagg atcaaaagtg tgaaatggga tttgggatca 960

gatttgtgct ccttgtgttc ttgtgttgtt gttatcactg ttgtcgtccg ctgctgggtc 1020

ccctctgctc cagtctctgt cattgcgagt ctcttcgagg agatggtccc cggcattaag 1080

tccctgttgc ctcgagtttg gcgactgggg tcttgctcgg tcagggcaga tctatatatg 1140

catagcttgc ggcgtctgtg aatagtgctg tttatttctt acaccttctg acttgttttc 1200

ttctcttttg acatttggag gaagtgttga caactacata agtaatctaa taatcgatcc 1260

ccaacccaga cagacagaca gatgtcgttg tgtaaaacat gcaaatccgt aaatccgata 1320

atttcatacc cagcggtacc tgtccgtatt ttttcttcct cttttttatc ttatcgccac 1380

catcacctga ctggggcggg gaaaatacca gcaggccgtc actcaccgta attactcagt 1440

aatcctcggt gactcaaacc cac 1463

<210> 13

<211> 2740

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

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 2740

<210> 14

<211> 6044

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

ctttacgcgc tcgctgattg ttcgacaccc gtcgctatat tctctgttta attgcctcga 60

agcttggctc cgtctccatc cgaccagaag gggggaggat ctttttctta ttttattttc 120

gacaatagtg tctttaatat ttccgaagca accgaaaatc cacgggaaag ccgctgattg 180

actggacgcc atcttctttc cgtgtcgtgt ggtctcctac ccagctgttc gtttgcctgg 240

tcccttatac gccggatcta tagagcgatc tccgtttgtt gagcggaagg tacccccaag 300

gacgatcatc gaaccaccgt cttttgtcac gtccaacaac cctcctcgtt tcagcaaaat 360

cacctttatt tgcagtgtcc ggattcgctc atctgtctca attttacact ctcccgaaca 420

gaaaacgaga gtggatcgtc agctagccat cttgccaacc ttgtctctct gtagtccttc 480

acacgaggtc ggacctggac tcaaaccatc gtggatcttt gtctcggctg aggacaccac 540

cttattacga actagcaggg aaatatctca aataggctgc gtttactgac gaagcctctc 600

cctgtgctga agtcattggt ctgttaaggt tggtgccaac ccccccctcc ctcgcccttt 660

gccacagact ctgttggtgc cttgcatcac tctccttcgt gtccccttcc ctctgtactc 720

cttgcttctc caaggtcgtg cctttgccgc acaagatccc aaggaaaatt agggtccacg 780

atgggttccg tgcgcttgtc acctcccctt gtatggtttc cgtcactaaa cttccggatg 840

tttgttgtta acccgccccc gtcaaggtgt agactacctc gtttagtcgc ttagggaccg 900

tgaccttttt tgcatattag atttcaaaag acctcagaga cgcatctctt gtcttctcat 960

ctcacctggt aagtcttgct ggtctatgca gcgagccggt tcctcgcctt ttgggagggt 1020

tcttactcac tagttgagta gttcgtgccc cgacctagtc ctcatcatcc tgagggaaac 1080

ttgagacaat ttcgcttcca ggaacgccag aaaagcgaaa aggtcttctt ctcacttcga 1140

ccggctcttc tcgctcgctc ttatccagtg cctgttacgg atatctgcca tccaacctat 1200

ccgccgcgtg aggttaaaac agcgccttca gatggaagct ggtttcctat aggttgtcag 1260

aagcattcat cgacagtgtg gctcacctct ggtctctatc ctgcagccaa cctttcgtgg 1320

tttcaccctg aggcgttgga tcctgatcgc tgattcgaag cttttcgtgg catcttcctc 1380

gtccgggctg acagccactt cgtgtcgtca ttcgttttgt ctcggagagc tcgctagtaa 1440

ctttccgaca tcctatcgtt ttccaggata aattcgctgt cattcgcttt ttttaagctg 1500

ttgtgcttcg ttgcagtttc tgcaacacct tcaaactggc ttactcgaag acgcacggtg 1560

ttttggtgag tccgggtctt cctgctcgct gcccaattgt tgctttttgg tcccttccca 1620

ttccattttg tttttcgact ggatcagaat atctgattca tcactgtctc gccgagtgcc 1680

ccagccttgt ttttcctgca gtcacaccca ccttctcacc cgccgaacac tgaactgacc 1740

cgggggcgtt ttacagttca attccgagtt acaaacaccg ttaattctag acgagtcgtt 1800

ggatttttcg gatcctgaag agtcacagtt tctcggtgct ttttcagacg gccttctggc 1860

caataaaatc cgccgcctcc accatttgta gaaaaatgtg acgaactcgt gagctctgta 1920

cagtgaccgg tgactctttc tggcatgcgg agagacggac ggacgcagag agaagggctg 1980

agtaataagc cactggccag acagctctgg cggctctgag gtgcagtgga tgattattaa 2040

tccgggaccg gccgcccctc cgccccgaag tggaaaggct ggtgtgcccc tcgttgacca 2100

agaatctatt gcatcatcgg agaatatgga gcttcatcga atcaccggca gtaagcgaag 2160

gagaatgtga agccaggggt gtatagccgt cggcgaaata gcatgccatt aacctaggta 2220

cagaagtcca attgcttccg atctggtaaa agattcacga gatagtacct tctccgaagt 2280

aggtagagcg agtacccggc gcgtaagctc cctaattggc ccatccggca tctgtagggc 2340

gtccaaatat cgtgcctctc ctgctttgcc cggtgtatga aaccggaaag gccgctcagg 2400

agctggccag cggcgcagac cgggaacaca agctggcagt cgacccatcc ggtgctctgc 2460

actcgacctg ctgaggtccc tcagtccctg gtaggcagct ttgccccgtc tgtccgcccg 2520

gtgtgtcggc ggggttgaca aggtcgttgc gtcagtccaa catttgttgc catattttcc 2580

tgctctcccc accagctgct cttttctttt ctctttcttt tcccatcttc agtatattca 2640

tcttcccatc caagaacctt tatttcccct aagtaagtac tttgctacat ccatactcca 2700

tccttcccat cccttattcc tttgaacctt tcagttcgag ctttcccact tcatcgcagc 2760

ttgactaaca gctaccccgc ttgagcagac atcaccatgc ctgaactcac cgcgacgtct 2820

gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag 2880

ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta 2940

aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc 3000

gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc 3060

atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct 3120

gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg 3180

agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc 3240

atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc 3300

agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa 3360

gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc 3420

ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc 3480

aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag 3540

cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt 3600

cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag 3660

ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc 3720

cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac 3780

cgacgcccca gcactcgtcc gagggcaaag gaatagagta gatgccgacc gcgggatcca 3840

cttaacgtta ctgaaatcat caaacagctt gacgaatctg gatataagat cgttggtgtc 3900

gatgtcagct ccggagttga gacaaatggt gttcaggatc tcgataagat acgttcattt 3960

gtccaagcag caaagagtgc cttctagtga tttaatagct ccatgtcaac aagaataaaa 4020

cgcgttttcg ggtttacctc ttccagatac agctcatctg caatgcatta atgcattgac 4080

tgcaacctag taacgccttn caggctccgg cgaagagaag aatagcttag cagagctatt 4140

ttcattttcg ggagacgaga tcaagcagat caacggtcgt caagagacct acgagactga 4200

ggaatccgct cttggctcca cgcgactata tatttgtctc taattgtact ttgacatgct 4260

cctcttcttt actctgatag cttgactatg aaaattccgt caccagcncc tgggttcgca 4320

aagataattg catgtttctt ccttgaactc tcaagcctac aggacacaca ttcatcgtag 4380

gtataaacct cgaaatcant tcctactaag atggtataca atagtaacca tgcatggttg 4440

cctagtgaat gctccgtaac acccaatacg ccggccgaaa cttttttaca actctcctat 4500

gagtcgttta cccagaatgc acaggtacac ttgtttagag gtaatccttc tttctagaag 4560

tcctcgtgta ctgtgtaagc gcccactcca catctccact cgagagaaga gcgggaggcg 4620

aagcgtcggt tggtccaatt cacacggatg caaagcggca gtactatcca cgcagatttc 4680

aagcctgtgg cgcctgaaga tcgtgctccc aatagcatct gcatcagctg catctactgg 4740

gatgggaaag acgaatgctt tgttaccagt gabandnsac cagccctatt cttccgcctt 4800

ggccgcgcct tactcgtacc cttctgtctg tcagcagcag agccaactcg ggctcacagc 4860

caccgtgaac agcagaaatt gggacttgaa tcctcttctc aactctgctg cagccactgg 4920

caatcccagc agcagcgcgt gctacaacta ccttgctccc atgccgtatt ctgtgcatgg 4980

ccaggcccac gggtcttgaa atacctgcgc gttttggttc ctccatggta atctgcatcg 5040

actggcgttt ggctttcccg ctttactgcg ttgtgatttc ttgcataatt tttgatacct 5100

ctttgattat cgtcactcct taaccgcgag attttggcac tgagccctgt ggattttcta 5160

gcacaggata actcagtcac gggcctcacg gactttaccg tttatttttc atgatcactg 5220

tgaatgatct catgtctctc cttataccat cgcccttctt catgccttaa tgcccgatat 5280

cctttttttt ctttgacgca ttatagacga actgttgctg ttcttcacca tttacaccat 5340

gtttttattt tttgtggtta atgttgcatt ttcatgtggg catgaggctt tcgctccttt 5400

acgaagctgt acaactatct ggcatagcat ggcgctgccg tcgttccttc taggttgcag 5460

gtttgggata acacttggga ttgatgtacc agacatctgg gttggcgaag gatcaaaagt 5520

gtgaaatggg atttgggatc agatttgtgc tccttgtgtt cttgtgttgt tgttatcact 5580

gttgtcgtcc gctgctgggt cccctctgct ccagtctctg tcattgcgag tctcttcgag 5640

gagatggtcc ccggcattaa gtccctgttg cctcgagttt ggcgactggg gtcttgctcg 5700

gtcagggcag atctatatat gcatagcttg cggcgtctgt gaatagtgct gtttatttct 5760

tacaccttct gacttgtttt cttctctttt gacatttgga ggaagtgttg acaactacat 5820

aagtaatcta ataatcgatc cccaacccag acagacagac agatgtcgtt gtgtaaaaca 5880

tgcaaatccg taaatccgat aatttcatac ccagcggtac ctgtccgtat tttttcttcc 5940

tcttttttat cttatcgcca ccatcacctg actggggcgg ggaaaatacc agcaggccgt 6000

cactcaccgt aattactcag taatcctcgg tgactcaaac ccac 6044

<210> 15

<211> 15

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

atgcctgaac tcacc 15

<210> 16

<211> 16

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

gagggcaaag gaatag 16

<210> 17

<211> 6756

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

gaattccctt gtatctctac acacaggctc aaatcaataa gaagaacggt tcgtcttttt 60

cgtttatatc ttgcatcgtc ccaaagctat tggcgggata ttctgtttgc agttggctga 120

cttgaagtaa tctctgcaga tctttcgaca ctgaaatacg tcgagcctgc tccgcttgga 180

agcggcgagg agcctcgtcc tgtcacaact accaacatgg agtacgataa gggccagttc 240

cgccagctca ttaagagcca gttcatgggc gttggcatga tggccgtcat gcatctgtac 300

ttcaagtaca ccaacgctct tctgatccag tcgatcatcc gctgaaggcg ctttcgaatc 360

tggttaagat ccacgtcttc gggaagccag cgactggtga cctccagcgt ccctttaagg 420

ctgccaacag ctttctcagc cagggccagc ccaagaccga caaggcctcc ctccagaacg 480

ccgagaagaa ctggaggggt ggtgtcaagg aggagtaagc tccttattga agtcggagga 540

cggagcggtg tcaagaggat attcttcgac tctgtattat agataagatg atgaggaatt 600

ggaggtagca tagcttcatt tggatttgct ttccaggctg agactctagc ttggagcata 660

gagggtcctt tggctttcaa tattctcaag tatctcgagt ttgaacttat tccctgtgaa 720

ccttttattc accaatgagc attggaatga acatgaatct gaggactgca atcgccatga 780

ggttttcgaa atacatccgg atgtcgaagg cttggggcac ctgcgttggt tgaatttaga 840

acgtggcact attgatcatc cgatagctct gcaaagggcg ttgcacaatg caagtcaaac 900

gttgctagca gttccaggtg gaatgttatg atgagcattg tattaaatca ggagatatag 960

catgatctct agttagctca ccacaaaagt cagacggcgt aaccaaaagt cacacaacac 1020

aagctgtaag gatttcggca cggctacgga agacggagaa gccaccttca gtggactcga 1080

gtaccattta attctatttg tgtttgatcg agacctaata cagcccctac aacgaccatc 1140

aaagtcgtat agctaccagt gaggaagtgg actcaaatcg acttcagcaa catctcctgg 1200

ataaacttta agcctaaact atacagaata agataggtgg agagcttata ccgagctccc 1260

aaatctgtcc agatcatggt tgaccggtgc ctggatcttc ctatagaatc atccttattc 1320

gttgacctag ctgattctgg agtgacccag agggtcatga cttgagccta aaatccgccg 1380

cctccaccat ttgtagaaaa atgtgacgaa ctcgtgagct ctgtacagtg accggtgact 1440

ctttctggca tgcggagaga cggacggacg cagagagaag ggctgagtaa taagccactg 1500

gccagacagc tctggcggct ctgaggtgca gtggatgatt attaatccgg gaccggccgc 1560

ccctccgccc cgaagtggaa aggctggtgt gcccctcgtt gaccaagaat ctattgcatc 1620

atcggagaat atggagcttc atcgaatcac cggcagtaag cgaaggagaa tgtgaagcca 1680

ggggtgtata gccgtcggcg aaatagcatg ccattaacct aggtacagaa gtccaattgc 1740

ttccgatctg gtaaaagatt cacgagatag taccttctcc gaagtaggta gagcgagtac 1800

ccggcgcgta agctccctaa ttggcccatc cggcatctgt agggcgtcca aatatcgtgc 1860

ctctcctgct ttgcccggtg tatgaaaccg gaaaggccgc tcaggagctg gccagcggcg 1920

cagaccggga acacaagctg gcagtcgacc catccggtgc tctgcactcg acctgctgag 1980

gtccctcagt ccctggtagg cagctttgcc ccgtctgtcc gcccggtgtg tcggcggggt 2040

tgacaaggtc gttgcgtcag tccaacattt gttgccatat tttcctgctc tccccaccag 2100

ctgctctttt cttttctctt tcttttccca tcttcagtat attcatcttc ccatccaaga 2160

acctttattt cccctaagta agtactttgc tacatccata ctccatcctt cccatccctt 2220

attcctttga acctttcagt tcgagctttc ccacttcatc gcagcttgac taacagctac 2280

cccgcttgag cagacatcac catgcctgaa ctcaccgcga cgtctgtcga gaagtttctg 2340

atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt 2400

gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat 2460

ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg 2520

gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca 2580

cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc 2640

gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca 2700

ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct 2760

gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg 2820

caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg 2880

cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt 2940

gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg 3000

aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag 3060

cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat 3120

cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca 3180

atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc 3240

gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact 3300

cgtccgaggg caaaggaata gagtagatgc cgaccgcggg atccacttaa cgttactgaa 3360

atcatcaaac agcttgacga atctggatat aagatcgttg gtgtcgatgt cagctccgga 3420

gttgagacaa atggtgttca ggatctcgat aagatacgtt catttgtcca agcagcaaag 3480

agtgccttct agtgatttaa tagctccatg tcaacaagaa taaaacgcgt tttcgggttt 3540

acctcttcca gatacagctc atctgcaatg cattaatgca ttgactgcaa cctagtaacg 3600

ccttncaggc tccggcgaag agaagaatag cttagcagag ctattttcat tttcgggaga 3660

cgagatcaag cagatcaacg gtcgtcaaga gacctacgag actgaggaat ccgctcttgg 3720

ctccacgcga ctatatattt gtctctaatt gtactttgac atgctcctct tctttactct 3780

gatagcttga ctatgaaaat tccgtcacca gcncctgggt tcgcaaagat aattgcatgt 3840

ttcttccttg aactctcaag cctacaggac acacattcat cgtaggtata aacctcgaaa 3900

tcanttccta ctaagatggt atacaatagt aaccatgcat ggttgcctag tgaatgctcc 3960

gtaacaccca atacgccggc cgaaactttt ttacaactct cctatgagtc gtttacccag 4020

aatgcacagg tacacttgtt tagaggtaat ccttctttct agaagtcctc gtgtactgtg 4080

taagcgccca ctccacatct ccactcgacc tgcaggcatg caagcttggc actggccgtc 4140

gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca 4200

catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa 4260

cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg 4320

tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag 4380

ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc 4440

ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt 4500

tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag 4560

gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg 4620

cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 4680

caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 4740

ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 4800

gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 4860

gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 4920

atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg 4980

caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 5040

gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 5100

accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 5160

ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 5220

gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca 5280

acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 5340

atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 5400

ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca 5460

gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 5520

gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 5580

tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 5640

taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 5700

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 5760

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 5820

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 5880

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 5940

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 6000

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 6060

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 6120

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 6180

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 6240

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 6300

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 6360

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 6420

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 6480

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc 6540

aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc 6600

gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca 6660

ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa 6720

caatttcaca caggaaacag ctatgaccat gattac 6756

Claims (6)

1. The application of Aspergillus niger genetically engineered bacteria in immobilized fermentation to produce citric acid is characterized in that chitin synthase transcription regulation gene MedA in the strain is inactivated; the original Aspergillus niger is Aspergillus Niger ATCC12846; the nucleotide sequence of the chitin synthase transcription regulation gene MedA inactivation is shown as SEQ ID NO. 2.

2. The use according to claim 1, wherein the immobilized fermentation uses a porous fibrous material as an immobilization carrier.

3. The use according to claim 2, wherein the immobilization support is pretreated by alkali soaking and acid soaking in sequence.

4. The use according to claim 1, wherein the amount of immobilized carrier used in the immobilized fermentation is 0.5-3g/L fermentation medium.

5. The use according to claim 1, wherein in the fermentation, the fermentation medium is prepared by: incubating the aqueous corn flour solution with a liquefying enzyme at 63-72deg.C for 38-43min; heating to 88-102 deg.C, and performing enzymolysis with liquefying enzyme until iodine solution does not change blue to obtain corn flour liquefied solution; filtering to obtain clear corn liquid; and uniformly mixing the obtained corn clear liquid with the corn flour liquefaction liquid to obtain the corn flour liquefaction liquid.

6. The use according to claim 1, wherein the fermentation conditions are: the fermentation temperature is 32-37 ℃, the fermentation time is 74-117h, and the fermentation rotating speed is 220-350rpm.

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