CN113817616A - Aspergillus niger genetically engineered bacterium with chitin synthase transcriptional regulation gene MedA inactivated and application thereof - Google Patents

Abstract

The invention discloses an aspergillus niger genetically engineered bacterium with a chitin synthase transcription regulation gene MedA inactivated and application thereof. The invention constructs an Aspergillus niger genetically engineered bacterium with a chitin synthase transcriptional regulatory gene MedA 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 of citric acid, shortening the fermentation period, and effectively solving the problems of low yield and long fermentation period in the existing industrial production of citric acid by aspergillus niger through fermentation.

Description

Aspergillus niger genetically engineered bacterium with chitin synthase transcriptional regulation gene MedA inactivated 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 with a chitin synthase transcription regulation gene MedA inactivated, a construction method and application thereof.

Background

Citric acid is used as the first major acid in biochemical products, is widely applied to industries such as food, medicine, daily chemicals and the like, and is one of the most widely used organic acids with the largest worldwide demand at present. China is the main country for producing citric acid, and the annual output accounts for 53 percent of the total world output. At present, the domestic citric acid production strain is mainly filamentous fungus Aspergillus niger, and the substrate is mainly grain crude processing products such as cassava powder, corn powder and the like. In recent years, due to the price of food in China, the labor cost generally rises, and the production cost of citric acid greatly rises. However, the rising cost makes the domestic citric acid market more and more difficult due to the competitive nature of the citric acid products in the international market.

In recent decades, the industrial production of citric acid has mainly been based on the liquid submerged fermentation mode. However, the liquid submerged fermentation has the natural defects of poor tolerance, easy aging, short catalytic aging and the like, so that the proliferation and differentiation capacity of cells of the aspergillus niger gradually decline, the autolysis phenomenon is serious, and the aspergillus niger cannot be continuously used for a long time, thereby causing the accumulation of a large amount of thallus residues in the fermentation process, and the problems of nutrient substance waste and low substrate conversion rate. It is also one of the major bottlenecks of the current production technology of the citric acid industry. The cell immobilization fermentation technology based on the biological membrane can continuously perform fermentation, shorten the production period, improve the yield, greatly improve the efficiency of citric acid fermentation and reduce the cost. Under the environment, cells can be gathered in large scale and high density, and the method has the characteristics of intelligent characteristics of self proliferation and self repair, ecological and efficient heat transfer system and long-term continuous reaction process. The idea and the method overcome the defects that the cells are fixed on the carrier by adopting methods such as polyvinyl alcohol gel embedding, glutaraldehyde crosslinking and the like in the prior art, and simultaneously overcome the problems of serious autolysis phenomenon of deep liquid fermentation cells in a liquid phase and limited increase of thallus density. For filamentous fungi, the adsorption method is simple to operate, only has physical action and no chemical change in the fixing process, can be theoretically used for cell fixing of all filamentous fungi, and is favorable for industrial large-scale application.

Disclosure of Invention

The purpose of the invention is as follows: 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 technical problem to be solved by the invention is to provide the construction method of the aspergillus niger genetically engineered bacteria.

The invention further aims to solve the technical problem of providing the application of the Aspergillus niger genetically engineered bacterium in citric acid production.

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

The Aspergillus niger genetically engineered bacterium is a genetically engineered bacterium which replaces partial sequence of gene MedA by hyg resistance gene (hygromycin resistance gene) through a double exchange method, and transcription regulation gene MedA can activate expression of chitin synthase A, B, C gene in Aspergillus niger to generate chitin which is secreted to the outside of cells and is an important component of extracellular matrix. The gene MedA is knocked out, so that the gene chitin synthase A, B, C cannot be activated, and the synthesis of chitin is inhibited, so that the formation of an Aspergillus niger extracellular matrix is reduced, an Aspergillus niger can not form excessive biological membranes in the immobilization process to influence oxygen transfer and mass transfer, and the immobilization fermentation effect is remarkably improved.

Among them, original Aspergillus Niger was Aspergillus Niger ATCC 12846.

Wherein, the inactivated nucleotide sequence of the chitin synthase transcription regulation gene MedA is shown as SEQ ID NO. 2, and the inactivated nucleotide sequence of the chitin synthase transcription regulation gene MedA 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 the genomic DNA of original Aspergillus niger ATCC 12846;

(2) amplifying to obtain an upstream homology arm and a downstream homology arm of a gene MedA 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 a gene MedA 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 chitin synthase transcriptional regulatory gene MedA inactivated.

In the step (2), 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 MedA is amplified; when the downstream homologous 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 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.

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 an application of the aspergillus niger genetically engineered bacterium in the production of citric acid by fermentation, wherein the aspergillus niger genetically engineered bacterium is used as a fermentation strain to prepare the citric acid by immobilized fermentation.

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

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

Wherein the immobilization carrier is cut into the same size and the volume is 0.5cm³A cube-shaped block.

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

Wherein, in the fermentation, the preparation method of the fermentation medium comprises the following steps: keeping the temperature of the corn flour water solution at 63-72 ℃ for 38-43min by using liquefying enzyme; heating to 88-102 deg.C, and performing enzymolysis with liquefying enzyme until iodine solution is unchanged blue to obtain liquefied semen Maydis powder; filtering to obtain corn clear liquid; mixing the obtained corn clear liquid and the corn flour liquefied liquid uniformly to obtain the corn liquid; preferably, the 220-280g/L corn flour water solution is subjected to heat preservation for 38-43min at the temperature of 63-72 ℃ by liquefying enzyme (0.5-1.5mL/L corn flour water solution); heating to 88-102 deg.C, and performing enzymolysis with liquefying enzyme (0.6-1.5mL/L corn flour water solution) until iodine solution is not changed into blue to obtain corn flour liquefied solution; filtering to obtain corn clear liquid; and (3) uniformly mixing the obtained corn clear liquid and the corn powder liquefied liquid according to the volume ratio of 100 (4-8) to obtain the corn-flour-based liquefied liquid.

Wherein the liquefying enzyme contains alpha-amylase, and the enzyme activity of the alpha-amylase is 72000-78000U/mL (1mL of enzyme solution is pH5.5 and at 85 ℃, the enzyme amount required for 1min hydrolysis of 1mg of soluble starch is 1 enzyme activity unit, U/mL).

Wherein the fermentation is to scrape the activated aspergillus niger genetically engineered bacterium plate with a spore scraping solution to prepare a spore solution, inoculate the spore solution in a fermentation culture medium containing an immobilized carrier, and ferment to obtain the citric acid.

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

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

Has the advantages 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 transcriptional regulatory gene MedA 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 of citric acid, shortening the fermentation period, and effectively solving the problems of low yield and long fermentation period in the existing industrial production of citric acid by aspergillus niger through fermentation.

Drawings

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

FIG. 1 is an electrophoretogram of the genome of Aspergillus Niger ATCC12846, wherein M is DNAmarker of DL10000, and lane 1 is the Aspergillus Niger genome.

FIG. 2 is a PAN7-1 plasmid map.

Fig. 3 is a PCR electrophoresis diagram of the upstream homology arm and the downstream homology arm of the MedA gene, wherein M is DNAMarker of DL5000, lane 1 is the upper homology arm of MedA, and lane 2 is the lower homology arm of MedA.

FIG. 4 shows the hyg expression element, where M is DL5000 DNAmarker and lane 1 is the hyg expression element.

FIG. 5 shows an electrophoretogram of knock-out fragment, wherein M is DL10000 DNAmarker, and lane 1 shows knock-out fragment.

FIG. 6 is a graph showing crystal violet staining.

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

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

FIG. 9 shows the difference between the original Aspergillus niger and Aspergillus niger genetically engineered bacteria 8 after continuous fermentation in batches.

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

Detailed Description

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

Example 1 construction of MedA knock-out bacterium of Aspergillus niger iron acquisition regulatory gene

Extraction of original Aspergillus niger genome

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

1. inoculating 1mL of scraped Aspergillus niger ATCC12846 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.

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

3. To the product obtained in step 2, 62.5. mu.L of Buffer KAC was added and mixed well. The mixture was kept on ice for 5min and centrifuged at 12000rpm for 5 min. The supernatant was collected at 600. mu.L, added to 600. mu.L of Buffer GB, and mixed well.

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

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

7. Repeat step 6 once.

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

9. The separation column in step 8 was placed on a new 1.5mL centrifuge tube, and 40. mu.L of sterilized water at 65 ℃ was added to the center of the separation column membrane, and 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 being DL10000 and No. 1 is extracted A.niger genome.

(II) amplifying upstream and downstream homologous arms of gene MedA 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 MedA-up-F as an upper primer and using MedA-up-R as a lower primer (the nucleotide sequence of the MedA-up-F is shown as SEQ ID NO:3, and the nucleotide sequence of the MedA-up-R is shown as SEQ ID NO: 4); the downstream homology arm is amplified by taking 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 10 s; (2) annealing at 55 ℃ for 30 s; (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 is finished, the PCR product is quantified by agarose gel electrophoresis, as shown in FIG. 3, M is DNA Marker of DL5000, No. 1 is the upper homologous arm of MedA, and No. 2 is the lower homologous arm of 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 resistance expression element is amplified by taking PAN7-1 plasmid (stored in a laboratory, the PAN7-1 plasmid map is shown in figure 2, and the nucleotide sequence is shown in SEQ ID NO:17) as a template, MedA-hyg-F as an upper primer, and MedA-hyg-R as a 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 10 s; (2) annealing at 55 ℃ for 30 s; (3) extending at 72 ℃ for 5min, and repeating the steps (1) to (3) 35 times. After the reaction, the PCR product was quantified by agarose gel electrophoresis, as shown in FIG. 4, where M is DNA Marker of DL5000 and No. 1 is hyg resistance expression element. Wherein the nucleotide sequence of the hyg resistance expression element is shown as SEQ ID NO. 13.

(IV) amplification of knockout fragment

The MedA gene knockout fragment is amplified by using a MedA upstream homology arm, a MedA downstream homology arm and a hyg resistance expression element as templates, MedA-F as an upper primer, MedA-R as a lower primer (the nucleotide sequence of MedA-F is shown as SEQ ID NO:9, and the nucleotide sequence of MedA-R is shown as SEQ ID NO: 10) and using an 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 95 ℃ for 10 s; (2) annealing at 55 ℃ for 30 s; (3) extending at 72 ℃ for 5min, and repeating the steps (1) to (3) 35 times. After the reaction, the PCR product was quantified by agarose gel electrophoresis, as shown in FIG. 5, M is DL10000 DNA Marker, and No. 1 is knockout fragment. The nucleotide sequence of the gene knockout fragment is shown as SEQ ID NO. 14.

(V) preparation and transformation of Aspergillus niger protoplast

1. Aspergillus niger ATCC12846 was inoculated into PDA plates and filled with spores, 3mL of the scrape spore buffer was added to the plates, the spores were scraped off with a spreading stick 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 1cm³Adding water 600mL into small pieces, boiling, maintaining for 30min, filtering with 4 layers of gauze to obtain potato juice, metering to 1L with 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 into 50mL of DP culture medium, culturing at 35 deg.C and 200rpm for 13-16h, and observing spore germination state by microscopic examination.

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

Enzymatic hydrolysates (lyase Lysing enzyme (Sigma L1412), crashease Driselase (Meclin D861435), snailase (Shanghai worker A600870) each 0.1g/10mL, cellulase (Sigma C2605) 400. mu.L/10 mL) were prepared and sterilized by filtration using sterile syringe filters.

4. Adding 2g of mycelium into the enzymolysis 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 of solution 5 was added, and the mixture was mixed by gun-suction to obtain protoplasts. Wherein the solution 5 is prepared by a self-prepared formula: KCl 4.47g, CaCl₂0.735g, MOP 0.2093g, adjusted to pH 6.0 with KOH and made up to 100mL with 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 50. mu.L of solution 4 is added, mixed evenly, and placed on ice for 15-30 min. Wherein the solution 4 is prepared by a self-made method, and the formula is as follows: PEG 800025 g, CaCl₂1.47g, KCl 4.47g, 10mM Tris, pH 7.5 adjusted with hydrochloric acid and made up to 100mL with water.

After 7.20min, 900 μ L of solution 4 was added, turned upside down several times and mixed, and left at room temperature 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 150mmol/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 shows that the transformation is successful, and a delta MedA strain is obtained.

Example 2 Crystal Violet staining experiment

Respectively inoculating original Aspergillus niger (ATCC12846) and genetically engineered bacteria (delta MedA strains) to a PDA (personal digital assistant) plate, adding 3mL of a sporulation buffer solution into the plate after spores grow, scraping the spores by using a coating rod, transferring the spores into a sterilized 5mL centrifuge tube, and performing constant volume to 2mL by using the sporulation buffer solution to obtain a spore solution. Quantitated and diluted to 10 using a hemocytometer⁴one/mL, followed by further dilutionTo 10³/mL，10²/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 for 72h at 35 ℃ 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. FIG. 6 and FIG. 7 show the difference between the crystal violet staining pattern and the OD value.

Wherein 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. 6 and 7 show that the.DELTA.MedA strain after decolorization was markedly lighter in purple color than the original strain, at 10²The color of the delta MedA 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 after the chitin synthase regulatory gene delta MedA is inactivated, the biological membrane is reduced.

Example 3 immobilized fermentation experiment of genetically engineered bacteria

1. Preparation of porous fiber material immobilized medium

Soaking a polyurethane material (with the molecular weight of 3000-. Cutting into 0.5cm³The vectors with the same size are arranged on the left and the 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 the corn flour aqueous solution in a 75 ℃ water bath kettle, 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 ℃, and liquefying for 60min until iodine solution does not turn blue to obtain corn flour liquefied solution; filtering the corn powder liquid to obtain a clear corn powder liquid; adding unfiltered corn flour liquefied liquid into the corn flour clear liquid as a feed back (the volume ratio of the unfiltered corn flour liquid to the corn clear liquid is 8 percent, and uniformly mixing the unfiltered corn flour liquid and the corn clear liquid to obtain a fermentation culture medium). 100mL of the homogenized fermentation medium was dispensed into 500mL Erlenmeyer flasks containing 0.2g of vector. Sterilizing and cooling for later use.

3. Immobilized continuous fermentation

(1) Inoculating the frozen Aspergillus niger genetically engineered bacteria and original Aspergillus niger spores onto a PDA (personal digital assistant) plate, culturing for 4-5 days at 35 ℃ in a constant-temperature incubator 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 first batch of immobilized fermentation is finished. Wherein the citric acid is determined by NaOH titration method and the total sugar is determined by DNS method.

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

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.

Wherein, the DNS method is as follows:

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 plotted on the ordinate, and the absorbance OD540 is plotted on the abscissa to prepare a standard curve.

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.

Determination of citric acid by high performance liquid chromatography:

and (3) preparing a standard curve: measuring the content of citric acid by high performance liquid chromatography, preparing a series of citric acid standard solutions with concentration of 0-10g/L, sucking 1mL of 0.22 μm membrane filter, and loading into a liquid phase vial for measurement. The analysis conditions were as follows: a RID difference detector; the mobile phase is 5mM H2SO 4; aminex HPX-87H organic acid column from Bio-Rad at a flow rate of 0.6 mL/min; the sample amount is 20 μ L, the column temperature is 55 deg.C, and the stop time is 20 min. The peak areas of the samples at different concentrations were determined. And (4) taking the concentration of the standard solution as a vertical coordinate, and taking the corresponding peak area as a horizontal coordinate to draw a standard curve.

The measurement method of the sample comprises the following steps: the supernatant was diluted appropriately to take 1ml, filtered through a 0.22 μm filter and placed in a liquid phase vial for assay. The measured peak area is taken into a standard curve to calculate the concentration.

FIG. 8 shows the difference between the Δ MedA strain and the original strain under fermentation conditions, and the results show that after 96 hours of fermentation, the yield of citric acid produced by fermentation of the Δ MedA strain 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 is improved by 12.5 percent compared with the original strain. Fig. 9 shows the difference in production intensity of Δ MedA strain and the original strain after 8 batches of continuous fermentation. Meanwhile, the cycle of knock-out bacteria is reduced from 96h to 60h, which is reduced by 37.5%. The fermentation strength is improved from 1.68g/L/h to 3.03g/L/h, and is improved by 80.3 percent. As can be seen from the figure, the fermentation intensity of the Δ MedA strain is not obviously reduced after 8 batches of continuous fermentation, while the fermentation intensity of the original strain is reduced by 30.5 percent after 8 batches of continuous fermentation, which shows that the continuous fermentation performance of the Δ MedA strain is obviously superior to that of the original strain. Meanwhile, as can be seen in Table 3, the total content of the heteropolyacid in the Δ MedA strain immobilized fermentation broth is reduced to 2.9g/L from 4.8g/L of the original strain, which is reduced by 39.6%. Compared with other genetic engineering bacteria, the content of the heteropolyacid is also obviously reduced by 19.4%. After the MedA gene is knocked out, the amount of bacteria adsorbed on the carrier is further reduced, so that the oxygen and mass transfer effects in the pore diameter of the carrier are obviously enhanced, byproducts such as oxalic acid and malic acid generated in a severe fermentation environment and in an anoxic state are reduced, and the quality of a finished citric acid product is obviously improved. Meanwhile, compared with the original strain, the yield, the conversion rate and the production intensity of the delta MedA strain are obviously improved, multi-batch continuous fermentation can be realized, the production intensity is not obviously reduced, so that great progress is made on the basis of producing citric acid by immobilized fermentation of aspergillus niger, and the method is suitable for industrial production.

TABLE 3 heteropolyacids in the fermentation broths

Wherein, the carrier after immobilized fermentation for 4 days is taken out, and adsorbed hyphae are washed off by 3 times of PBS washing. As shown in FIG. 10, the carrier with mycelia adhered thereto had a large amount of original bacteria, the pore diameter of the carrier was almost filled, and the amount of the knockout bacteria was moderate.

The invention provides an aspergillus niger genetically engineered bacterium with a chitin synthase transcription regulation gene MedA inactivated and a thinking and a method for application thereof, and a plurality of methods and ways for realizing the technical scheme are provided. All the components not specified in the present embodiment can be realized by the prior art.

Sequence listing

<110> Nanjing university of industry

COFCO Biotechnology Co.,Ltd.

<120> Aspergillus niger genetically engineered bacterium with inactivated chitin synthase transcriptional regulatory gene MedA and application thereof

<160> 17

<170> SIPOSequenceListing 1.0

<210> 1

<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 (10)

1. An Aspergillus niger genetically engineered bacterium is characterized in that a chitin synthase transcription regulation gene MedA 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 chitin synthase transcription regulation gene MedA is shown in SEQ ID NO 2.

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

5. The application of claim 4, wherein the Aspergillus niger genetically engineered bacterium is used as a fermentation strain to prepare the citric acid through immobilized fermentation.

6. The use according to claim 5, wherein the immobilized fermentation uses porous fiber material as an immobilized carrier.

7. The use according to claim 6, wherein the immobilized carrier is pretreated by alkali soaking and acid soaking in sequence.

8. The use according to claim 5, wherein the immobilized carrier is used in an amount of 0.5-3g/L fermentation medium in the immobilized fermentation.

9. The use according to claim 4, wherein in the fermentation, the fermentation medium is prepared by: keeping the temperature of the corn flour water solution at 63-72 ℃ for 38-43min by using liquefying enzyme; heating to 88-102 deg.C, and performing enzymolysis with liquefying enzyme until iodine solution is unchanged blue to obtain liquefied semen Maydis powder; filtering to obtain corn clear liquid; and mixing the obtained corn clear liquid and the corn powder liquefied liquid uniformly to obtain the corn liquid.

10. The use according to claim 4, wherein the fermentation conditions are: the fermentation temperature is 32-37 ℃, the fermentation time is 74-117h, and the fermentation rotation speed is 220-350 rpm.

Images