CN114292761B - A genetically engineered strain of Aspergillus niger and its construction method and application - Google Patents

Abstract

The invention discloses an aspergillus niger genetically engineered bacterium, a construction method and application thereof, wherein the recombinant aspergillus niger genetically engineered bacterium is obtained by obtaining a regulatory gene AcuK through over-expressing iron in a starting bacterium. The invention constructs a recombinant aspergillus niger genetically engineered bacterium for obtaining and controlling gene AcuK over-expression by gene knockout means. The recombinant genetically engineered bacterium can increase the amount of the biological film of aspergillus niger in the process of producing citric acid by immobilized fermentation, simultaneously lighten the phenomenon of carrier agglomeration, exert the advantage of immobilized fermentation, improve the yield of the citric acid and shorten the fermentation period.

Description

Aspergillus niger genetically engineered bacterium, construction method and application

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to an aspergillus niger genetic engineering bacterium for obtaining regulatory gene AcuK from iron, and a construction method and application thereof.

Background

Because of its wide application, citric acid plays an important role in industries such as medicine, food, agriculture, etc., and consumes a large amount of citric acid each year. Among the markets for organic acids, citric acid has a market share of up to 70%. In the food industry, citric acid is widely used for producing various beverages, candies, biscuits and other foods, and can also be used as a flavoring agent or an antioxidant, and citrate plays an important role in the food industry; in the pharmaceutical industry, citrate can help the rapid dissolution of active ingredients in the drug or be used as an anticoagulant; in the chemical industry, citric acid is used in large amounts as a complexing agent, a masking agent, a builder, etc.; citric acid is also widely used in textile industry, animal husbandry, environmental protection, and the like. In the current industrial production of citric acid, aspergillus niger biological fermentation is one of the most important ways, and the main raw materials are grain crops such as corn. However, the current grain reserves in China cannot meet the national demands, the grain price is generally increased, and the production cost of citric acid is increased, so that the citric acid fermentation method needs to be improved, and the fermentation cost is reduced.

The cell immobilized fermentation technology developed based on the immobilized enzyme technology can enable 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. At present, there are four main methods for cell immobilization: embedding, crosslinking, covalent bonding, and adsorption. The adsorption method is based on the interaction force of the cell surface and the carrier surface, mainly hydrophobic force and electrostatic force to promote the adsorption of cells on the carrier surface. 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 a recombinant 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 recombinant Aspergillus niger genetically engineered bacterium.

The invention also solves the technical problem of providing the recombinant bacterial agent.

The invention further aims to provide the recombinant aspergillus niger genetically engineered bacterium and application of the microbial inoculum.

The invention finally aims to provide a method for producing citric acid.

In order to solve the first technical problem, the invention discloses a recombinant aspergillus niger genetically engineered bacterium, which is used for obtaining a regulatory gene AcuK by over-expressing iron in a starting bacterium.

Wherein the starting bacterium is Aspergillus niger Aspergillus Niger ATCC12846.

Wherein, the nucleotide sequence of the overexpression iron acquisition regulatory gene AcuK is shown as SEQ ID NO. 1.

The method for obtaining the regulatory gene AcuK through over-expression of iron in the starting strain is to introduce a plasmid of the regulatory gene AcuK through over-expression of iron into the starting strain, so that the expression level of the AcuK gene in the aspergillus niger is increased, the gene AcuK can inhibit SheaA gene in the aspergillus niger and induce the expression of HapX gene, thereby stimulating the assimilation of reduced iron and promoting the iron absorption mediated by siderophores. After the AcuK gene is over-expressed, the increase of intracellular ferric ion level promotes the formation of hydrophobin, the hydrophobic acting force is enhanced, so that the hydrophobicity of the spore surface is obviously enhanced, the formation of a biological film is increased, and meanwhile, the over-expressed AcuK gene also changes the phenotype of the biological film adsorbed on the carrier, so that the film generated on the carrier forms a filiform structure, the phenomenon of agglomeration is reduced, the mass transfer efficiency is improved, and the effects of immobilized fermentation and citric acid production are affected.

Wherein, the nucleotide sequence of the plasmid of the iron overexpression acquisition regulatory gene AcuK is shown as SEQ ID NO. 2.

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

(1) Extracting genome DNA of original aspergillus niger;

(2) Amplifying the genome DNA obtained in the step (1) serving as a template to obtain a gene AcuK; amplifying to obtain a GpdA promoter gene and a TrpC terminator gene by taking a plasmid PAN7-1 as a template; the gene AcuK, gpdA promoter gene and TrpC terminator are used as templates, and the gene overexpression fragments are obtained through overlap extension PCR amplification;

(3) The PAN7-1 plasmid was double digested with XbaI and HindIII fast-cutting enzymes to give a linearized PAN7-1 plasmid.

(4) And (3) connecting the gene overexpression fragment obtained in the step (2) with the linearized plasmid PAN7-1 by a one-step cloning method to obtain the overexpression plasmid PAN7-1-AcuK.

(5) Introducing the over-expression plasmid PAN7-1-AcuK into Aspergillus niger protoplast, and carrying out homologous recombination to obtain the Aspergillus niger genetically engineered bacterium of which the over-expression iron is used for obtaining the regulatory gene AcuK.

In the step (1), the original aspergillus niger is aspergillus niger Aspergillus Niger ATCC12846.

In the step (2), the nucleotide sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4 are used as primers when the gene AcuK is amplified; amplifying GpdA promoter gene with the nucleotide sequences shown in SEQ ID No. 5 and SEQ ID No. 6 as primers; when the TrpC terminator gene is amplified, the nucleotide sequences shown in SEQ ID NO. 7 and SEQ ID NO. 8 are used as primers; the overlapping extension PCR amplified gene is over-expressed by using the nucleotide sequences shown in SEQ ID NO. 9 and SEQ ID NO. 10 as primers.

In the step (2), the nucleotide sequence of the GpdA promoter gene is shown as SEQ ID NO. 11, the nucleotide sequence of the TrpC terminator gene is shown as SEQ ID NO. 12, and the nucleotide sequence of the gene over-expression fragment is shown as SEQ ID NO. 13.

In the step (4), the nucleotide sequence of the over-expression plasmid PAN7-1-AcuK is shown as SEQ ID NO. 2.

In order to solve the third technical problem, the invention discloses a microbial inoculum which contains the recombinant aspergillus niger genetically engineered bacterium described in the first technical problem.

In order to solve the fourth technical problem, the invention discloses the recombinant Aspergillus niger genetically engineered bacterium of the first technical problem and application of the microbial inoculum of the third technical problem in citric acid production by fermentation.

Wherein the fermentation is to take polyurethane material as an immobilization medium for immobilization fermentation.

Wherein the temperature of the fermentation is 30-37 ℃.

Wherein the fermentation time is 72-115 h.

Wherein the rotating speed of the fermentation is 200-330 rpm.

In order to solve the fifth technical problem, the invention discloses a method for producing citric acid, which comprises the step of fermenting and culturing the recombinant aspergillus niger genetically engineered bacterium described in the first technical problem to obtain the citric acid.

Wherein the fermentation is carried out by taking the recombinant aspergillus niger genetically engineered bacteria as a fermentation strain and taking a polyurethane material as an immobilization medium.

Specifically, the method for producing citric acid comprises the following steps:

s1, preparation of an immobilization carrier: soaking the immobilized carrier in 0.5-2M NaOH for 30min-2h, washing with water until the pH is neutral, soaking in 0.5-2 MHCl for 30min-2h, washing with water until the pH is neutral, and drying to obtain the pretreated immobilized carrier; cutting the pretreated large immobilized carrier into the same size with the volume of 0.5cm ³ Is of (3)A square block carrier;

s2, fermentation: scraping the activated recombinant aspergillus niger genetically engineered bacteria flat plate by using a spore scraping liquid to prepare spore liquid; inoculating the strain into sterilized fermentation medium containing immobilized carrier at 0.4% (v/v), and immobilized fermenting to obtain citric acid.

In step S1, the preparation of the immobilized carrier is preferably to soak the immobilized carrier with 1M NaOH for 1h, rinse with water to neutral pH, soak in 1M HCl for 1h, rinse with water to neutral pH, and dry to obtain the pretreated immobilized carrier.

In the step S2, the preparation steps of the fermentation medium for immobilized fermentation are as follows:

a. weighing corn flour, adding water and preparing into corn flour solution with the concentration of 200-300 g/L; adding 0.5-1 mL of liquefying enzyme into each 1L of corn flour liquid, and preserving heat for 35-45 min at 60-75 ℃ to obtain corn flour liquid;

b. heating the corn flour liquid to 85-105 ℃, adding 0.5-1 mL of liquefying enzyme into each 1L of corn flour liquid, carrying out enzymolysis for 45-60 min until the iodine liquid does not change blue, and filtering the corn flour liquid to obtain corn clear liquid;

c. adding the unfiltered corn flour liquid obtained in the step b into the corn clear liquid obtained in the step b, wherein the volume ratio of the unfiltered corn flour liquid to the corn clear liquid is (2-10): 100, and uniformly mixing to obtain the immobilized fermentation medium.

Wherein the liquefying enzyme contains alphA-Amylase, the enzyme activity of the alphA-Amylase is 70000-80000U/mL (the enzyme amount required for hydrolyzing 1mg of soluble starch for 1min under the conditions of pH5.5 and temperature of 85 ℃ is 1 enzyme activity unit, U/mL).

In the step S2, the content of the immobilized carrier is 2g/L of fermentation medium.

In the step S2, the fermentation temperature is 30-37 ℃.

In the step S2, the fermentation time is 72-115 hours.

In the step S2, the rotating speed of the fermentation is 200-330 rpm.

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

the invention constructs a recombinant aspergillus niger genetically engineered bacterium for obtaining and controlling gene AcuK over-expression by gene knockout means. The recombinant genetically engineered bacterium can increase the amount of the biological film of aspergillus niger in the process of producing citric acid by immobilized fermentation, simultaneously lighten the phenomenon of carrier agglomeration, exert the advantage of immobilized fermentation, improve the yield of the citric acid and shorten the fermentation period.

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 genome of A.niger Aspergillus Niger ATCC12846.

FIG. 2 is a PAN7-1 plasmid map.

FIG. 3 is a PCR electrophoretogram of the GpdA promoter gene, the TrpC terminator gene and the Acuk gene, wherein M1 is DNAMaroer of DL1000, lane 1 is the GpdA promoter gene, lane 2 is the TrpC terminator gene, lane 3 is the Acuk gene and M2 is DNAMaroer of DL 5000.

FIG. 4 is an electrophoretogram of a gene over-expression fragment, wherein M is marker lane, and 1 is a gene over-expression fragment.

FIG. 5 is a crystal violet staining chart.

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

FIG. 7 is a morphology of the original 24-well plate film.

FIG. 8 is a diagram of the morphology of the 24-well plate membrane of the +pAcuK strain.

FIG. 9 shows fermentation results of original Aspergillus niger and Aspergillus niger genetically engineered bacteria.

FIG. 10 results of continuous batch fermentation of original Aspergillus niger and Aspergillus niger genetically engineered bacteria.

FIG. 11 is a biological membrane electron microscope image of the original A.niger.

FIG. 12 is a diagram of a biological membrane electron microscope of 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 AcuK overexpression Strain of Aspergillus niger iron acquisition regulatory Gene

(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 Aspergillus Niger ATCC12846 spore liquid is inoculated into 50mL of DP culture medium, and cultured for 24h 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 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 tube with 500 mu L Buffer HS II added in advance, mixing uniformly, adding 10 mu L RNase A, shaking sufficiently, mixing uniformly, and bathing in a water bath at 56 ℃ for 10min.

3. 62.5. Mu.L Buffer KAC was added to step 2 and thoroughly mixed. 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. Placing Spin Column in Collection Tube, transferring the solution into Spin Column, centrifuging at 12000rpm for 1min, and discarding the filtrate;

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

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

7. repeating the step 6 once;

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

9. the Spin Column was placed on a new 1.5mL centrifuge tube, 40. Mu.L of 65℃sterilized water was added to the center of the Spin Column membrane, and the mixture was allowed to stand at room temperature for 1min. The DNA was eluted by centrifugation at 12000rpm for 2 min. Determination of Aspergillus niger genome concentration by agarose gel electrophoresis As shown in FIG. 1, DNA marker with M of DL15000, both No. 1 and No. 2 were the extracted Aspergillus niger genome.

(II) PCR technique is used to amplify GpdA promoter and TrpC terminator

Amplifying the GpdA promoter gene by taking a PAN7-1 plasmid (stored in a laboratory, wherein the map of the PAN7-1 plasmid is shown in FIG. 2, the nucleotide sequence is shown in SEQ ID NO: 16), gpdA-F as an upper primer, and GpdA-R as a lower primer (the nucleotide sequence of GpdA-F is shown in SEQ ID NO:5, and the nucleotide sequence of GpdA-R is shown in SEQ ID NO: 6); the TrpC-F is used as an upper primer and the TrpC-R is used as a lower primer (the nucleotide sequence of the TrpC-F is shown as SEQ ID NO:7, and the nucleotide sequence of the TrpC-R is shown as 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.

TABLE 1 PCR reaction System and reaction conditions

After completion of the reaction, the PCR products were quantified by agarose gel electrophoresis, see FIG. 3. The nucleotide sequence of the GpdA promoter gene is shown as SEQ ID NO. 11, and the nucleotide sequence of the TrpC terminator gene is shown as SEQ ID NO. 12.

And (III) amplifying the over-expressed gene AcuK.

Amplifying the over-expressed gene AcuK by taking the original Aspergillus niger genome extracted in the step (I) as a template, acuK-F as an upper primer and AcuK-R as a lower primer (the nucleotide sequence of the AcuK-F is shown as SEQ ID NO:3 and the nucleotide sequence of the AcuK-R is shown as SEQ ID NO: 4). 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 completion of the reaction, the PCR products were quantified by agarose gel electrophoresis, see FIG. 3. Wherein the nucleotide sequence of the over-expressed gene AcuK is shown as SEQ ID NO. 1. FIG. 3 shows the DNA Marker with M DL5000, the GpdA promoter gene No. 1, the TrpC terminator gene No. 2 and the overexpressed gene AcuK No. 3.

(IV) amplified Gene overexpression fragment

The GpdA promoter gene, the TrpC terminator gene and the overexpressed gene AcuK are used as templates, gpdA-AcuM-F is used as an upper primer, acuM-TrpC-R is used as a lower primer (the nucleotide sequence of GpdA-AcuM-F is shown as SEQ ID NO:9, the nucleotide sequence of AcuM-TrpC-R is shown as SEQ ID NO: 10), and the overlapping extension PCR (Overlap PCR) technology is used for amplifying the overexpressed fragment of the AcuK gene. The reaction system is shown in 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 completion of the reaction, the PCR products were quantified by agarose gel electrophoresis, see FIG. 4. The nucleotide sequence of the gene over-expression fragment is shown in SEQ ID NO. 13. FIG. 4 shows that M is a DNA Marker of DL10000 and No. 1 is a gene overexpression fragment.

(V) digestion of plasmid Pan7-1 with restriction enzyme XbaI and HindIII

The cleavage reaction system shown in Table 2 was prepared on ice, reacted at 37℃for 30min, and then the fragments were recovered after confirmation of the correctness by gel running.

TABLE 2 cleavage reaction System

And (six) connecting the gene over-expression fragment and the digested plasmid Pan7-1 to obtain a recombinant plasmid PAN7-1-AcuK with a nucleotide sequence shown in SEQ ID NO. 2

The enzyme-linked system shown in Table 3 was prepared on ice, and immediately after 30min in a 37℃water bath, the ice was incubated for five minutes and kept at-20℃until use.

TABLE 3 enzyme-linked reaction system

(seventh) transformation of recombinant plasmid PAN7-1-AcuK into E.coli T1

1. Taking a 200. Mu.L E.coli T1 competent cell (Beijing full gold Biotechnology Co., ltd. CD 501-03) suspension from a-80℃refrigerator, thawing at room temperature, and immediately placing on ice after thawing;

2. adding the recombinant plasmid PAN7-1-AcuK obtained in the step (six) into E.coli T1 competent cell suspension, gently shaking, and standing on ice for 30min;

heat shock in water bath at a temperature of 3.42 ℃ for 45-90s, and immediately cooling on ice for 5min;

4. adding 1mL of LB liquid medium, mixing uniformly, and carrying out shaking culture at 37 ℃ for 40min to recover the thallus to a normal growth state;

5. shaking the bacterial liquid uniformly, taking 100 mu L of the bacterial liquid, coating the bacterial liquid on an LB resistance plate, inverting a culture dish, and culturing for 12 hours in a constant temperature incubator at 37 ℃; wherein the formulation of the LB-resistant plate is as follows: 10g/L of sodium chloride, 10g/L of tryptone, 5g/L of yeast powder and 20g/L of agar powder, sterilizing for 20min at 121 ℃, and adding ampicillin to a final concentration of 50 mu g/mL after cooling;

6. and (3) picking single colonies from the resistance plate, after bacterial protection, extracting plasmid PAN7-1-AcuK according to the step (III), and performing agarose gel electrophoresis verification and sequencing verification on the extracted plasmids.

Preparation and transformation of Aspergillus niger protoplast

1. Original Aspergillus niger was inoculated onto PDA plates, spores were grown, 3mL of a spore scraping buffer was added to the plates, spores were scraped off with a coating 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.

Enzymatic hydrolysate (lytic enzyme Arg-L1412, crashing enzyme Driselase Arg-D9515, snailase Snail-Arg-A600870 each 0.1g/10mL, cellulase sigma C2730400. Mu.L/10 mL) was prepared and sterilized by filtration through a sterile syringe.

4. Adding 2g of mycelium into enzyme solution, 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 solutin 5 was added, and the mixture was blown and mixed with a gun to obtain protoplasts. Wherein Solution5 is self-made, and the formula is as follows: 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 the protoplast was pipetted into a 1.5mL sterile centrifuge tube, and 10. Mu.L of the PAN7-1-AcuK constructed in step (seven) was added and mixed with it. Then 50. Mu.L of Solution4 was added, mixed well, placed on ice and timed for 15-30min. Wherein Solution4 is self-made 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 Solution4 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 JP-F and JP-R (the nucleotide sequence of JP-F is shown as SEQ ID NO:14, the nucleotide sequence of JP-R is shown as SEQ ID NO: 15), agarose electrophoresis shows amplified bands, which indicate successful transformation, and the recombinant Aspergillus niger genetically engineered bacteria are obtained: +pacuk strain.

Example 2 Crystal Violet staining experiments

Inoculating original Aspergillus niger and genetically engineered bacteria to PDA plate, adding 3mL of spore scraping buffer solution to the plate after spore grows, scraping off spores with a coating rod, transferring to sterilized 5mL centrifugeIn the tube, the spore liquid was obtained by constant volume to 2mL with the curettage buffer. Quantitated by a hemocytometer and diluted to 10 ⁵ individual/mL, followed by continued dilution to 10 ⁴ /mL，10 ³ /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 36 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. 5 and 6. In addition, changes in biofilm morphology at the bottom of the 24-well plate were seen, see fig. 7 and 8.

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

The results in FIGS. 5 and 6 show that the +pAcuK strain was significantly darker in purple than the original strain after decolorization, and the OD values detected by the microplate reader, the data were consistent with the color. Indicating that the biological film is increased after the iron acquisition regulatory gene AcuK is over-expressed. The results in FIGS. 7 and 8 show that +pAcuK strains formed more hyphae on the biofilm morphology at the bottom of the well plate than on the surface of the original biofilm.

Example 3 immobilized fermentation experiment of genetically engineered bacteria

1. Preparation of porous fibrous material immobilization medium

The polyurethane material is soaked in NaOH of 1M for 1h, washed by pure water, soaked in HCl of 1M for 1h, then washed by pure water until the pH value is neutral, and then put into a drying oven of 65 ℃ to be dried 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/L of corn flour water solution, placing the corn flour water solution in a 75 ℃ water bath kettle, adding 1mL of liquefying enzyme (60000U/L) when the corn flour solution reaches 65 ℃, liquefying for 40min, heating the water bath kettle to 95 ℃, adding 1mL of liquefying enzyme when the corn flour solution reaches 85 ℃, liquefying for 60min until the iodine solution does not change blue, filtering the corn flour solution to obtain corn flour clear liquid, adding unfiltered corn flour solution as a return material (the volume ratio of the unfiltered corn flour solution to the corn clear liquid is 8%), and uniformly mixing. Each 100mL was dispensed into 500mL Erlenmeyer flasks containing 2g/L of carrier. Sterilizing and cooling for standby.

3. 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 fermentation is ended. Wherein, the citric acid is measured by NaOH titration and the total sugar is measured by DNS method.

Wherein,,

the NaOH titration method comprises the following steps: 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.

The DNS method is as follows: a) 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. b) 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, adding 0.5mL of each sugar standard solution into 15mL of centrifuge tubes, adding 0.5mL of DNS solution into each centrifuge tube, placing the centrifuge tubes into a boiling water bath for reaction for 5min, placing the centrifuge tubes into ice water for cooling, adding 8mL of pure water into each centrifuge tube, mixing uniformly, and measuring the absorbance value OD540 at the wavelength of 540nm, wherein 0.0g/L is used as a reference. The concentration of the standard solution is on the ordinate, and the absorbance OD540 is on the abscissa, and a standard curve is drawn. c. ) 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.

FIG. 9 shows the difference between the +pAcuK strain and the original strain under fermentation conditions, showing that the average yield of citric acid produced by fermentation of the +pAcuK strain after 96 hours of fermentation is 186.3g/L and the average yield of citric acid produced by fermentation of the original strain is 161.2g/L. In terms of the conversion rate, the conversion rate of the +pAcuK strain reaches 103.5 percent, which is improved by 13.9 percent compared with the original strain, and the period is also reduced from 84 hours to 72 hours. FIG. 10 shows the difference in production intensity between the +pAcuK strain and the original strain after 12 batches of continuous fermentation. As can be seen from the graph, the fermentation strength of the +pAcuK strain is not obviously reduced after 12 batches of continuous fermentation, and the fermentation strength of the original strain after 12 batches of continuous fermentation is reduced by 31.5%, which shows that the continuous fermentation performance of the +pAcuK strain is obviously superior to that of the original strain. Aspergillus niger (Aspergillus Niger ATCC 12846) is a high-yield citric acid strain, the yield can be improved on the basis, the period is obviously shortened, the continuous fermentation effect is also obviously enhanced, so that the method can be said to be greatly improved on the basis of immobilized fermentation of Aspergillus niger into citric acid, and the method is suitable for industrial production.

(3) The carrier after 4d of immobilized fermentation was taken out, and the adsorbed mycelia were washed off by washing 3 times with PBS. As shown in FIG. 11 and FIG. 12, the amount of bacteria on the carrier of the +pAcuK strain was significantly increased as compared with that of the original bacteria.

(4) The mycelium-attached carrier is dried and then weighed, and the weight of the original carrier is subtracted to obtain the weight of the attached film. The original strain had a membrane content of 4.48g/L and the +pAcuK strain had a membrane content of 6.81g/L, which was increased by 52.0%.

(5) Filtering the culture medium with filter cloth to separate mycelium, oven drying, weighing, and adding the weight of the membrane to obtain total mycelium content in the fermentation system. The mycelium weight of the original strain was 28.42g/L, and the mycelium weight of the +pAcuK strain was 24.87g/L. The total hypha content in the fermentation system of the + pAcuK strain was found to be reduced by 15.76% and the adsorption of hypha was found to be increased by 11.68%. The mycelium is less in favor of improving the fermentation yield under the condition of the same sugar content in the culture medium.

The invention provides an aspergillus niger genetically engineered bacterium, a construction method and an application thought and method, and a method for realizing the technical scheme is more than one method and way, 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 skilled in the art without departing from the principle of the invention, and the improvements and modifications are also considered 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

<120> A strain of Aspergillus niger genetically engineered bacterium, construction method and application thereof

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2844

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

gaaaacactt cggcgcattg caacagtcag tctcactttc tgactgctgc tgactgagca 60

cacgcaaggt ccttcgtggt cgccgaccct cctactatct atctaccacc accactacgc 120

cagccgattc ctagccgtcg tcgtgtggat tctatgcgcc cgaccggcca acgcccgatt 180

tctttgcgac tcccagagta aggcctggtg aggttgatca gtggactctc gacaaagatc 240

tccttctgtt gtgcgctggt tcgcaacgcg acaaaaggca gtcagtcttg ctcaaaattt 300

acggtggcaa ccccacgcgc tcgcccgcct tgcagctgct tcactcaccc tgtcgctgat 360

cgcctcgatt cacgacgcgg agtactaccc ggttagacgg attgataacg gccgtatcgc 420

acacacatac acctcggtta caattgtttg ccgatatgaa cgcggaaccg aaggagcaag 480

attccccagc tccgtcggct gagcgcacgg aagccagtca ggagattagt gctgccggag 540

cacaggccga caagccgaag accgaggcca acggcgacgg gactgcaaat ggtgcctccg 600

cgaacggcca gaagcctaac ccgaaagacc cgtcgagacc ccggaggaag aaggcgagac 660

gggcgtgttt tgcttgccag cgcgcacact tgacttgcgg tacgactgac cttacttcca 720

gcgttgctta cattcaacct tttactctcc aattacacat gcgtttagcg cccatacatg 780

cgcactctag cggcgcatgt cttctgcgcg tctgactgaa cttgcaaact aatcgaggtt 840

tcgcataggt gatgagcgac cgtgtcagcg gtgcattaag cgcgggctcc aggatgcgtg 900

tcacgacggg gtccggaaga aggccaagta cctccacgac gcgccggatg gagcgttgat 960

gccggggatt ggcgggacgt tttacaacaa ccccatgcgc aacagcttgc ccctctctcg 1020

gaatggcgcc aatgccgtga acgcgaccgg tcagcagagc gctggcgcaa acttctatcc 1080

tactccccag tcgactacct atgtctacca agagaatacc atcaaccagg gctcatttcc 1140

ttcgcaatcg cctgtgtcgc cgacattcaa cctcaaggcc acgcctacgg ctcgaaccaa 1200

cagcctctcc tccgtcaacc cgcaaccacc gtccacgagc gtctccggac ccccaggcca 1260

gggtcaaaac ccgttcgccg gtcccttctt tgaccccagc gaccccgctc tcttcaactt 1320

tgacctttcc agcatgaact ttgagaaccg atacggtgcg ttagagtttg gcatgctggg 1380

acatatggcg acgggcgcgg gcgattcacc gagtgattcg gccacgcagc gcgggtctat 1440

ggggcgcagt ggctcggcac aatatgcttc aactcccatc actggggcac caggctttgg 1500

ggaaagcccg ggcaaccagc agccatttat gttcggggac ccgcttctca acgaatggcc 1560

cagcgggcag gccccgggac aaccgcatct gccgggggtt tatccgcagt ccgggcaagg 1620

cagcgcgatt cctggtcatc tgagcaaagc ggatgcgccg catgcatttg ccatcgaaag 1680

cggaccggcc agtttcaata gccccggtgc gacgactagt ccgcagatga cgactggact 1740

cgaagagacc ccgtttcaca gtgcggtggc aagcaagtcg aacgggttag ccccgcacgg 1800

acaacgaccg atgattacaa caccgagctt gaagcatcag aacctgcagg tcggcgtgcg 1860

gcgacgacaa cgtaacccgt cggccatcta cgacagcgtc aaggagccgt acgcatacac 1920

cagccgcttc catggcctca cggcattcat ccaacgacgg ttcccgccgc agaagacgct 1980

tcagatcgcc aaggcgctgg cgtcgatccg gccgtcgttc atcgcgacga ctaagacgct 2040

caatcgggac gacctgatct tcatggagaa gtgcttccag cggacgttgt gggaatacga 2100

ggactttatc aacgcgtgcg gcacgccgac catcgtgtgc cgtcggacgg gtgagattgc 2160

tgctgtgggc aaggagttca gtatcttgac gggatggaag aaggatgtac tgctcggcaa 2220

agagccgaat ctcaatgtca acacgggtgg ctcgtcaatg ccgaactccg gggcgtcatc 2280

acgcagcttc accccccggt cgacagtgga caacacgccc gggcgcccgc agccggtgtt 2340

cctggcagag ttgttggacg atgacagcgt ggtgcagttc tacgaggatt ttgcacggct 2400

ggcgtttggc gactcgcgcg gtagcgtgat gacaacgtgc aagctgttga agtacaagac 2460

gaaggaggac atggagggcg cagcagcgga ggatagccag cgctggaaca accacctgcg 2520

caagggaggg attgccagcg aggcggggat gaaccagcta gggttcaagg acggcaaggt 2580

cgaatgtgcg tactgctgga cagtgaagcg agatgtattc gacataccga tgttgattgt 2640

gatgaatgtg agtaatacca gcgttcccgt gctgtgatga tgatgctaat gagggtatag 2700

ttcttaccgt gcatttgatt tatgatcata attgtttcat gttcataccc gggccggata 2760

tcaaaaagga tgatttctga ttatttttac tgttagtttt tggcactgac cagatgcatc 2820

tggtcttgta ttggtacata ttgc 2844

<210> 2

<211> 9705

<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 ctagataaaa 2700

tccgccgcct ccaccatttg tagaaaaatg tgacgaactc gtgagctctg tacagtgacc 2760

ggtgactctt tctggcatgc ggagagacgg acggacgcag agagaagggc tgagtaataa 2820

gccactggcc agacagctct ggcggctctg aggtgcagtg gatgattatt aatccgggac 2880

cggccgcccc tccgccccga agtggaaagg ctggtgtgcc cctcgttgac caagaatcta 2940

ttgcatcatc ggagaatatg gagcttcatc gaatcaccgg cagtaagcga aggagaatgt 3000

gaagccaggg gtgtatagcc gtcggcgaaa tagcatgcca ttaacctagg tacagaagtc 3060

caattgcttc cgatctggta aaagattcac gagatagtac cttctccgaa gtaggtagag 3120

cgagtacccg gcgcgtaagc tccctaattg gcccatccgg catctgtagg gcgtccaaat 3180

atcgtgcctc tcctgctttg cccggtgtat gaaaccggaa aggccgctca ggagctggcc 3240

agcggcgcag accgggaaca caagctggca gtcgacccat ccggtgctct gcactcgacc 3300

tgctgaggtc cctcagtccc tggtaggcag ctttgccccg tctgtccgcc cggtgtgtcg 3360

gcggggttga caaggtcgtt gcgtcagtcc aacatttgtt gccatatttt cctgctctcc 3420

ccaccagctg ctcttttctt ttctctttct tttcccgaaa acacttcggc gcattgcaac 3480

agtcagtctc actttctgac tgctgctgac tgagcacacg caaggtcctt cgtggtcgcc 3540

gaccctccta ctatctatct accaccacca ctacgccagc cgattcctag ccgtcgtcgt 3600

gtggattcta tgcgcccgac cggccaacgc ccgatttctt tgcgactccc agagtaaggc 3660

ctggtgaggt tgatcagtgg actctcgaca aagatctcct tctgttgtgc gctggttcgc 3720

aacgcgacaa aaggcagtca gtcttgctca aaatttacgg tggcaacccc acgcgctcgc 3780

ccgccttgca gctgcttcac tcaccctgtc gctgatcgcc tcgattcacg acgcggagta 3840

ctacccggtt agacggattg ataacggccg tatcgcacac acatacacct cggttacaat 3900

tgtttgccga tatgaacgcg gaaccgaagg agcaagattc cccagctccg tcggctgagc 3960

gcacggaagc cagtcaggag attagtgctg ccggagcaca ggccgacaag ccgaagaccg 4020

aggccaacgg cgacgggact gcaaatggtg cctccgcgaa cggccagaag cctaacccga 4080

aagacccgtc gagaccccgg aggaagaagg cgagacgggc gtgttttgct tgccagcgcg 4140

cacacttgac ttgcggtacg actgacctta cttccagcgt tgcttacatt caacctttta 4200

ctctccaatt acacatgcgt ttagcgccca tacatgcgca ctctagcggc gcatgtcttc 4260

tgcgcgtctg actgaacttg caaactaatc gaggtttcgc ataggtgatg agcgaccgtg 4320

tcagcggtgc attaagcgcg ggctccagga tgcgtgtcac gacggggtcc ggaagaaggc 4380

caagtacctc cacgacgcgc cggatggagc gttgatgccg gggattggcg ggacgtttta 4440

caacaacccc atgcgcaaca gcttgcccct ctctcggaat ggcgccaatg ccgtgaacgc 4500

gaccggtcag cagagcgctg gcgcaaactt ctatcctact ccccagtcga ctacctatgt 4560

ctaccaagag aataccatca accagggctc atttccttcg caatcgcctg tgtcgccgac 4620

attcaacctc aaggccacgc ctacggctcg aaccaacagc ctctcctccg tcaacccgca 4680

accaccgtcc acgagcgtct ccggaccccc aggccagggt caaaacccgt tcgccggtcc 4740

cttctttgac cccagcgacc ccgctctctt caactttgac ctttccagca tgaactttga 4800

gaaccgatac ggtgcgttag agtttggcat gctgggacat atggcgacgg gcgcgggcga 4860

ttcaccgagt gattcggcca cgcagcgcgg gtctatgggg cgcagtggct cggcacaata 4920

tgcttcaact cccatcactg gggcaccagg ctttggggaa agcccgggca accagcagcc 4980

atttatgttc ggggacccgc ttctcaacga atggcccagc gggcaggccc cgggacaacc 5040

gcatctgccg ggggtttatc cgcagtccgg gcaaggcagc gcgattcctg gtcatctgag 5100

caaagcggat gcgccgcatg catttgccat cgaaagcgga ccggccagtt tcaatagccc 5160

cggtgcgacg actagtccgc agatgacgac tggactcgaa gagaccccgt ttcacagtgc 5220

ggtggcaagc aagtcgaacg ggttagcccc gcacggacaa cgaccgatga ttacaacacc 5280

gagcttgaag catcagaacc tgcaggtcgg cgtgcggcga cgacaacgta acccgtcggc 5340

catctacgac agcgtcaagg agccgtacgc atacaccagc cgcttccatg gcctcacggc 5400

attcatccaa cgacggttcc cgccgcagaa gacgcttcag atcgccaagg cgctggcgtc 5460

gatccggccg tcgttcatcg cgacgactaa gacgctcaat cgggacgacc tgatcttcat 5520

ggagaagtgc ttccagcgga cgttgtggga atacgaggac tttatcaacg cgtgcggcac 5580

gccgaccatc gtgtgccgtc ggacgggtga gattgctgct gtgggcaagg agttcagtat 5640

cttgacggga tggaagaagg atgtactgct cggcaaagag ccgaatctca atgtcaacac 5700

gggtggctcg tcaatgccga actccggggc gtcatcacgc agcttcaccc cccggtcgac 5760

agtggacaac acgcccgggc gcccgcagcc ggtgttcctg gcagagttgt tggacgatga 5820

cagcgtggtg cagttctacg aggattttgc acggctggcg tttggcgact cgcgcggtag 5880

cgtgatgaca acgtgcaagc tgttgaagta caagacgaag gaggacatgg agggcgcagc 5940

agcggaggat agccagcgct ggaacaacca cctgcgcaag ggagggattg ccagcgaggc 6000

ggggatgaac cagctagggt tcaaggacgg caaggtcgaa tgtgcgtact gctggacagt 6060

gaagcgagat gtattcgaca taccgatgtt gattgtgatg aatgtgagta ataccagcgt 6120

tcccgtgctg tgatgatgat gctaatgagg gtatagttct taccgtgcat ttgatttatg 6180

atcataattg tttcatgttc atacccgggc cggatatcaa aaaggatgat ttctgattat 6240

ttttactgtt agtttttggc actgaccaga tgcatctggt cttgtattgg tacatattgc 6300

ggatccactt aacgttactg aaatcatcaa acagcttgac gaatctggat ataagatcgt 6360

tggtgtcgat gtcagctccg gagttgagac aaatggtgtt caggatctcg ataagatacg 6420

ttcatttgtc caagcagcaa agagtgcctt ctagtgattt aatagctcca tgtcaacaag 6480

aataaaacgc gttttcgggt ttacctcttc cagatacagc tcatctgcaa tgcattaatg 6540

cattgactgc aacctagtaa cgccttncag gctccggcga agagaagaat agcttagcag 6600

agctattttc attttcggga gacgagatca agcagatcaa cggtcgtcaa gagacctacg 6660

agactgagga atccgctctt ggctccacgc gactatatat ttgtctctaa ttgtactttg 6720

acatgctcct cttctttact ctgatagctt gactatgaaa attccgtcac cagcncctgg 6780

gttcgcaaag ataattgcat gtttcttcct tgaactctca agcctacagg acacacattc 6840

atcgtaggta taaacctcga aatcanttcc tactaagatg gtatacaata gtaaccatgc 6900

atggttgcct agtgaatgct ccgtaacacc caatacgccg gccgaaactt ttttacaact 6960

ctcctatgag tcgtttaccc agaatgcaca ggtacacttg tttagaggta atccttcttt 7020

ctagaagtcc tcgtgtactg tgtaagcgcc cactccacat ctccactcga aagcttggca 7080

ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc 7140

cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc 7200

ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt 7260

acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat 7320

gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct 7380

tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt 7440

cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta 7500

tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg 7560

ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 7620

ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 7680

attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 7740

gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 7800

ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 7860

cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 7920

gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 7980

tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 8040

gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 8100

ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 8160

tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 8220

gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 8280

caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 8340

cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 8400

atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 8460

gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 8520

attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 8580

cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 8640

atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 8700

tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 8760

ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 8820

ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 8880

cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 8940

gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 9000

gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 9060

acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 9120

gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 9180

agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 9240

tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 9300

agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 9360

cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 9420

gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc 9480

ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac 9540

aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact 9600

cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg 9660

agcggataac aatttcacac aggaaacagc tatgaccatg attac 9705

<210> 3

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

tcttttctct ttcttttccc gaaaacactt cggcgcattg 40

<210> 4

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

cagtaacgtt aagtggatcc gcaatatgta ccaatacaag 40

<210> 5

<211> 36

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

gaggtaatcc ttctttctag ataaaatccg ccgcct 36

<210> 6

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

caatgcgccg aagtgttttc gggaaaagaa agagaaaaga 40

<210> 7

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

cttgtattgg tacatattgc ggatccactt aacgttactg 40

<210> 8

<211> 36

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

acgacggcca gtgccaagct ttcgagtgga gatgtg 36

<210> 9

<211> 36

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

gaggtaatcc ttctttctag ataaaatccg ccgcct 36

<210> 10

<211> 36

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

acgacggcca gtgccaagct ttcgagtgga gatgtg 36

<210> 11

<211> 761

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

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 c 761

<210> 12

<211> 725

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

ggatccactt aacgttactg aaatcatcaa acagcttgac gaatctggat ataagatcgt 60

tggtgtcgat gtcagctccg gagttgagac aaatggtgtt caggatctcg ataagatacg 120

ttcatttgtc caagcagcaa agagtgcctt ctagtgattt aatagctcca tgtcaacaag 180

aataaaacgc gttttcgggt ttacctcttc cagatacagc tcatctgcaa tgcattaatg 240

cattgactgc aacctagtaa cgccttncag gctccggcga agagaagaat agcttagcag 300

agctattttc attttcggga gacgagatca agcagatcaa cggtcgtcaa gagacctacg 360

agactgagga atccgctctt ggctccacgc gactatatat ttgtctctaa ttgtactttg 420

acatgctcct cttctttact ctgatagctt gactatgaaa attccgtcac cagcncctgg 480

gttcgcaaag ataattgcat gtttcttcct tgaactctca agcctacagg acacacattc 540

atcgtaggta taaacctcga aatcanttcc tactaagatg gtatacaata gtaaccatgc 600

atggttgcct agtgaatgct ccgtaacacc caatacgccg gccgaaactt ttttacaact 660

ctcctatgag tcgtttaccc agaatgcaca ggtacacttg tttagaggta atccttcttt 720

ctaga 725

<210> 13

<211> 4375

<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 cgaaaacact tcggcgcatt 780

gcaacagtca gtctcacttt ctgactgctg ctgactgagc acacgcaagg tccttcgtgg 840

tcgccgaccc tcctactatc tatctaccac caccactacg ccagccgatt cctagccgtc 900

gtcgtgtgga ttctatgcgc ccgaccggcc aacgcccgat ttctttgcga ctcccagagt 960

aaggcctggt gaggttgatc agtggactct cgacaaagat ctccttctgt tgtgcgctgg 1020

ttcgcaacgc gacaaaaggc agtcagtctt gctcaaaatt tacggtggca accccacgcg 1080

ctcgcccgcc ttgcagctgc ttcactcacc ctgtcgctga tcgcctcgat tcacgacgcg 1140

gagtactacc cggttagacg gattgataac ggccgtatcg cacacacata cacctcggtt 1200

acaattgttt gccgatatga acgcggaacc gaaggagcaa gattccccag ctccgtcggc 1260

tgagcgcacg gaagccagtc aggagattag tgctgccgga gcacaggccg acaagccgaa 1320

gaccgaggcc aacggcgacg ggactgcaaa tggtgcctcc gcgaacggcc agaagcctaa 1380

cccgaaagac ccgtcgagac cccggaggaa gaaggcgaga cgggcgtgtt ttgcttgcca 1440

gcgcgcacac ttgacttgcg gtacgactga ccttacttcc agcgttgctt acattcaacc 1500

ttttactctc caattacaca tgcgtttagc gcccatacat gcgcactcta gcggcgcatg 1560

tcttctgcgc gtctgactga acttgcaaac taatcgaggt ttcgcatagg tgatgagcga 1620

ccgtgtcagc ggtgcattaa gcgcgggctc caggatgcgt gtcacgacgg ggtccggaag 1680

aaggccaagt acctccacga cgcgccggat ggagcgttga tgccggggat tggcgggacg 1740

ttttacaaca accccatgcg caacagcttg cccctctctc ggaatggcgc caatgccgtg 1800

aacgcgaccg gtcagcagag cgctggcgca aacttctatc ctactcccca gtcgactacc 1860

tatgtctacc aagagaatac catcaaccag ggctcatttc cttcgcaatc gcctgtgtcg 1920

ccgacattca acctcaaggc cacgcctacg gctcgaacca acagcctctc ctccgtcaac 1980

ccgcaaccac cgtccacgag cgtctccgga cccccaggcc agggtcaaaa cccgttcgcc 2040

ggtcccttct ttgaccccag cgaccccgct ctcttcaact ttgacctttc cagcatgaac 2100

tttgagaacc gatacggtgc gttagagttt ggcatgctgg gacatatggc gacgggcgcg 2160

ggcgattcac cgagtgattc ggccacgcag cgcgggtcta tggggcgcag tggctcggca 2220

caatatgctt caactcccat cactggggca ccaggctttg gggaaagccc gggcaaccag 2280

cagccattta tgttcgggga cccgcttctc aacgaatggc ccagcgggca ggccccggga 2340

caaccgcatc tgccgggggt ttatccgcag tccgggcaag gcagcgcgat tcctggtcat 2400

ctgagcaaag cggatgcgcc gcatgcattt gccatcgaaa gcggaccggc cagtttcaat 2460

agccccggtg cgacgactag tccgcagatg acgactggac tcgaagagac cccgtttcac 2520

agtgcggtgg caagcaagtc gaacgggtta gccccgcacg gacaacgacc gatgattaca 2580

acaccgagct tgaagcatca gaacctgcag gtcggcgtgc ggcgacgaca acgtaacccg 2640

tcggccatct acgacagcgt caaggagccg tacgcataca ccagccgctt ccatggcctc 2700

acggcattca tccaacgacg gttcccgccg cagaagacgc ttcagatcgc caaggcgctg 2760

gcgtcgatcc ggccgtcgtt catcgcgacg actaagacgc tcaatcggga cgacctgatc 2820

ttcatggaga agtgcttcca gcggacgttg tgggaatacg aggactttat caacgcgtgc 2880

ggcacgccga ccatcgtgtg ccgtcggacg ggtgagattg ctgctgtggg caaggagttc 2940

agtatcttga cgggatggaa gaaggatgta ctgctcggca aagagccgaa tctcaatgtc 3000

aacacgggtg gctcgtcaat gccgaactcc ggggcgtcat cacgcagctt caccccccgg 3060

tcgacagtgg acaacacgcc cgggcgcccg cagccggtgt tcctggcaga gttgttggac 3120

gatgacagcg tggtgcagtt ctacgaggat tttgcacggc tggcgtttgg cgactcgcgc 3180

ggtagcgtga tgacaacgtg caagctgttg aagtacaaga cgaaggagga catggagggc 3240

gcagcagcgg aggatagcca gcgctggaac aaccacctgc gcaagggagg gattgccagc 3300

gaggcgggga tgaaccagct agggttcaag gacggcaagg tcgaatgtgc gtactgctgg 3360

acagtgaagc gagatgtatt cgacataccg atgttgattg tgatgaatgt gagtaatacc 3420

agcgttcccg tgctgtgatg atgatgctaa tgagggtata gttcttaccg tgcatttgat 3480

ttatgatcat aattgtttca tgttcatacc cgggccggat atcaaaaagg atgatttctg 3540

attattttta ctgttagttt ttggcactga ccagatgcat ctggtcttgt attggtacat 3600

attgcggatc cacttaacgt tactgaaatc atcaaacagc ttgacgaatc tggatataag 3660

atcgttggtg tcgatgtcag ctccggagtt gagacaaatg gtgttcagga tctcgataag 3720

atacgttcat ttgtccaagc agcaaagagt gccttctagt gatttaatag ctccatgtca 3780

acaagaataa aacgcgtttt cgggtttacc tcttccagat acagctcatc tgcaatgcat 3840

taatgcattg actgcaacct agtaacgcct tncaggctcc ggcgaagaga agaatagctt 3900

agcagagcta ttttcatttt cgggagacga gatcaagcag atcaacggtc gtcaagagac 3960

ctacgagact gaggaatccg ctcttggctc cacgcgacta tatatttgtc tctaattgta 4020

ctttgacatg ctcctcttct ttactctgat agcttgacta tgaaaattcc gtcaccagcn 4080

cctgggttcg caaagataat tgcatgtttc ttccttgaac tctcaagcct acaggacaca 4140

cattcatcgt aggtataaac ctcgaaatca nttcctacta agatggtata caatagtaac 4200

catgcatggt tgcctagtga atgctccgta acacccaata cgccggccga aactttttta 4260

caactctcct atgagtcgtt tacccagaat gcacaggtac acttgtttag aggtaatcct 4320

tctttctaga agtcctcgtg tactgtgtaa gcgcccactc cacatctcca ctcga 4375

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

tcgatgatgc agcttgggcg 20

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

gtactccgcg tcgtgaatcg 20

<210> 16

<211> 5388

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

taaaatccgc cgcctccacc atttgtagaa aaatgtgacg aactcgtgag ctctgtacag 60

tgaccggtga ctctttctgg catgcggaga gacggacgga cgcagagaga agggctgagt 120

aataagccac tggccagaca gctctggcgg ctctgaggtg cagtggatga ttattaatcc 180

gggaccggcc gcccctccgc cccgaagtgg aaaggctggt gtgcccctcg ttgaccaaga 240

atctattgca tcatcggaga atatggagct tcatcgaatc accggcagta agcgaaggag 300

aatgtgaagc caggggtgta tagccgtcgg cgaaatagca tgccattaac ctaggtacag 360

aagtccaatt gcttccgatc tggtaaaaga ttcacgagat agtaccttct ccgaagtagg 420

tagagcgagt acccggcgcg taagctccct aattggccca tccggcatct gtagggcgtc 480

caaatatcgt gcctctcctg ctttgcccgg tgtatgaaac cggaaaggcc gctcaggagc 540

tggccagcgg cgcagaccgg gaacacaagc tggcagtcga cccatccggt gctctgcact 600

cgacctgctg aggtccctca gtccctggta ggcagctttg ccccgtctgt ccgcccggtg 660

tgtcggcggg gttgacaagg tcgttgcgtc agtccaacat ttgttgccat attttcctgc 720

tctccccacc agctgctctt ttcttttctc tttcttttcc catcttcagt atattcatct 780

tcccatccaa gaacctttat ttcccctaag taagtacttt gctacatcca tactccatcc 840

ttcccatccc ttattccttt gaacctttca gttcgagctt tcccacttca tcgcagcttg 900

actaacagct accccgcttg agcagacatc accatgcctg aactcaccgc gacgtctgtc 960

gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc 1020

gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat 1080

agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg 1140

ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc 1200

tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt 1260

ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc 1320

gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata 1380

tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt 1440

gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc 1500

cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata 1560

acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac 1620

atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg 1680

aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt 1740

gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt 1800

cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc 1860

agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga 1920

cgccccagca ctcgtccgag ggcaaaggaa tagagtagat gccgaccgcg ggatccactt 1980

aacgttactg aaatcatcaa acagcttgac gaatctggat ataagatcgt tggtgtcgat 2040

gtcagctccg gagttgagac aaatggtgtt caggatctcg ataagatacg ttcatttgtc 2100

caagcagcaa agagtgcctt ctagtgattt aatagctcca tgtcaacaag aataaaacgc 2160

gttttcgggt ttacctcttc cagatacagc tcatctgcaa tgcattaatg cattgactgc 2220

aacctagtaa cgccttncag gctccggcga agagaagaat agcttagcag agctattttc 2280

attttcggga gacgagatca agcagatcaa cggtcgtcaa gagacctacg agactgagga 2340

atccgctctt ggctccacgc gactatatat ttgtctctaa ttgtactttg acatgctcct 2400

cttctttact ctgatagctt gactatgaaa attccgtcac cagcncctgg gttcgcaaag 2460

ataattgcat gtttcttcct tgaactctca agcctacagg acacacattc atcgtaggta 2520

taaacctcga aatcanttcc tactaagatg gtatacaata gtaaccatgc atggttgcct 2580

agtgaatgct ccgtaacacc caatacgccg gccgaaactt ttttacaact ctcctatgag 2640

tcgtttaccc agaatgcaca ggtacacttg tttagaggta atccttcttt ctagaagtcc 2700

tcgtgtactg tgtaagcgcc cactccacat ctccactcga cctgcaggca tgcaagcttg 2760

gcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat 2820

cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat 2880

cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc 2940

cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct 3000

gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg 3060

gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 3120

tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc 3180

ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt 3240

cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat 3300

ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg 3360

agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt 3420

tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga 3480

gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa 3540

gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt 3600

attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt 3660

gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc 3720

agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga 3780

ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat 3840

cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct 3900

gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc 3960

cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 4020

gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 4080

ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 4140

acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca 4200

ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta 4260

aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc 4320

aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa 4380

ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca 4440

ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta 4500

actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc 4560

caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca 4620

gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta 4680

ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag 4740

cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt 4800

cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc 4860

acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac 4920

ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac 4980

gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc 5040

tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat 5100

accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag 5160

cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac 5220

gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc 5280

actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt 5340

gtgagcggat aacaatttca cacaggaaac agctatgacc atgattac 5388

Claims (8)

1. A recombinant Aspergillus niger genetically engineered strain, characterized in that the iron acquisition regulatory gene AcuK is overexpressed in the starting strain to obtain a recombinant Aspergillus niger genetically engineered strain; the starting strain is Aspergillus Niger ATCC12846; the overexpression The nucleotide sequence of the iron acquisition regulatory gene AcuK is shown in SEQ ID NO: 1. 2. The recombinant Aspergillus niger genetically engineered strain according to claim 1, characterized in that the method of overexpressing the iron acquisition regulatory gene AcuK in the originating strain is to introduce a plasmid overexpressing the iron acquisition regulatory gene AcuK into the originating strain. 3. The recombinant Aspergillus niger genetically engineered strain according to claim 2, characterized in that the nucleotide sequence of the plasmid overexpressing the iron acquisition regulatory gene AcuK is shown in SEQ ID NO: 2. 4. A microbial agent, characterized by containing the recombinant Aspergillus niger genetically engineered bacterium according to any one of claims 1 to 3. 5. The recombinant Aspergillus niger genetically engineered strain described in any one of claims 1 to 3, or the application of the bacterial agent described in claim 4 in fermentation to produce citric acid. 6. A method for producing citric acid, characterized by comprising the step of fermenting and cultivating the recombinant Aspergillus niger genetically engineered strain according to any one of claims 1 to 4 to obtain citric acid. 7. The method for producing citric acid according to claim 6, or the application in producing citric acid through fermentation according to claim 5, characterized in that the fermentation is an immobilized fermentation using polyurethane material as an immobilized medium. 8. The method for producing citric acid according to claim 6, or the application in fermentation for producing citric acid according to claim 5, characterized in that the temperature of the fermentation is 30-37°C; the time of the fermentation is 72～115h.