CN112779170B - Recombinant aspergillus niger genetically engineered bacterium and construction method and application thereof - Google Patents

Abstract

The invention discloses a recombinant Aspergillus niger genetically engineered bacterium, a construction method and application thereof, wherein iron acquisition regulatory gene in the recombinant Aspergillus niger genetically engineered bacterium is inactivated. 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 between the carrier and the outside, thereby exerting the advantages of immobilized fermentation, improving the yield and sugar-acid conversion rate of citric acid, shortening the fermentation period, and effectively solving the problems of low yield and long fermentation period in the prior industrial production of citric acid by aspergillus niger through fermentation; and the obtained recombinant Aspergillus niger genetically engineered bacteria can be fermented continuously in 12 batches, and the fermentation intensity is not obviously reduced.

Description

Recombinant aspergillus niger genetically engineered bacterium and construction method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant aspergillus niger genetically engineered bacterium, a construction method and application thereof.

Background

Because of its wide use, citric acid plays an important role in the industries of medicine, food, agriculture, etc., and a large amount of citric acid is consumed every year. In the market for organic acids, citric acid has a market share as high as 70%. In the food industry, citric acid is commonly used for producing various beverages, candies, biscuits and other foods, can also be used as a flavoring agent or an antioxidant, and citrate also plays an important role in the food industry; in the pharmaceutical industry, citrate can help the quick dissolution of active ingredients in medicines or be used as an anticoagulant; in the chemical industry, citric acid is used in large quantities as complexing agents, masking agents, builders, etc.; citric acid is also widely used in textile industry, animal husbandry, environmental protection, and the like. At present, in the industrial production of citric acid, aspergillus niger is used for biological fermentation, which is one of the most main ways, and the main raw material of the citric acid is grain crops such as corn and the like. However, the current food storage in China cannot meet the national demand, the food price generally rises, and the production cost of citric acid also rises, so that the improvement of a citric acid fermentation method is needed to reduce the fermentation cost.

The cell immobilization fermentation technology developed based on the immobilized enzyme technology can continuously carry out fermentation, shorten the production period, improve the yield, greatly improve the fermentation efficiency of the citric acid and reduce the cost. At present, the cell immobilization method mainly comprises four methods: entrapment, cross-linking, covalent bonding, and adsorption. The adsorption method is based on the interaction force between the cell surface and the carrier surface, mainly hydrophobic force and electrostatic force to promote the adsorption of cells on the carrier surface. 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 technical problem to be solved by the invention is to provide a recombinant aspergillus niger genetically engineered bacterium aiming at the defects of the prior art.

The invention also provides a construction method of the recombinant aspergillus niger genetically engineered bacterium.

The invention further provides application of the recombinant Aspergillus niger genetically engineered bacterium.

The invention idea is as follows: according to the invention, a double exchange method is adopted, and a hygromycin resistance gene (hyg resistance gene) is used for replacing part of a sequence of an iron acquisition regulatory gene (AcuM gene) in the Aspergillus niger genetically engineered bacteria, so that the recombinant Aspergillus niger genetically engineered bacteria is obtained. Wherein, the AcuM gene can inhibit SreaA gene in Aspergillus niger and induce HapX gene expression, thereby stimulating reduced iron assimilation and promoting iron absorption mediated by a siderophore. After the AcuM gene is knocked out, the reduction of intracellular iron ion level inhibits the formation of hydrophobin, the hydrophobic action force is weakened, the hydrophobicity of the surface of spores is obviously weakened, and the formation of biofilm is reduced, so that the effects of immobilized fermentation and citric acid production are influenced.

In order to solve the first technical problem, the invention discloses a recombinant aspergillus niger genetically engineered bacterium, wherein AcuM gene in the recombinant aspergillus niger genetically engineered bacterium is inactivated.

Wherein the original Aspergillus Niger strain is Aspergillus Niger ATCC12846.

Wherein, the nucleotide sequence of the inactivated AcuM gene is shown in SEQ ID NO. 1.

Wherein, the inactivated nucleotide sequence of the AcuM gene is shown in SEQ ID NO. 2.

In order to solve the second technical problem, the invention discloses a method for constructing the recombinant aspergillus niger genetically engineered bacterium, which comprises the following steps:

(1) Amplifying to obtain an upstream homology arm and a downstream homology arm of an AcuM gene by taking the original genomic DNA of the Aspergillus niger as a template;

(2) Amplifying to obtain a hygromycin resistance gene by taking the plasmid PAN7-1 as a template;

(3) Taking the upstream homology arm and the downstream homology arm of the AcuM gene obtained in the step (1) and the hygromycin resistance gene obtained in the step (2) as templates, and performing overlap extension PCR amplification to obtain a gene knockout fragment;

(4) And (4) introducing the gene knockout fragment obtained in the step (3) into an Aspergillus niger protoplast for homologous recombination to obtain AcuM inactivated recombinant Aspergillus niger genetically engineered bacterium.

In step (1), the original mold genetic bacteria is preferably Aspergillus Niger ATCC12846.

In the step (1), the upstream homology arm of the AcuM gene is obtained by amplifying primers with nucleotide sequences of SEQ ID NO.3 and SEQ ID NO. 4.

In the step (1), the nucleotide sequence of the upstream homology arm of the AcuM gene is shown as SEQ ID NO. 11.

In the step (1), the downstream homology arm of the AcuM gene is obtained by amplifying primers with nucleotide sequences shown as SEQ ID NO.5 and SEQ ID NO. 6.

In the step (1), the nucleotide sequence of the downstream homology arm of the AcuM gene is shown as SEQ ID NO. 12.

In the step (2), the hygromycin resistance gene is obtained by amplifying primers with nucleotide sequences shown as SEQ ID NO.7 and SEQ ID NO. 8.

In the step (2), the nucleotide sequence of the hygromycin resistance gene is shown as SEQ ID NO. 13.

In the step (3), the gene knockout fragment is obtained by amplifying primers with nucleotide sequences shown as SEQ ID NO.9 and SEQ ID NO. 10.

In the step (3), the nucleotide sequence of the knockout fragment is shown as SEQ ID NO. 14.

In order to solve the third technical problem, the invention discloses application of the recombinant aspergillus niger genetically engineered bacterium in fermentation preparation of citric acid.

Preferably, the fermentation is immobilized fermentation, namely the recombinant aspergillus niger genetically engineered bacteria are used as fermentation strains to prepare the citric acid through immobilized fermentation, the hydrophobicity of the recombinant aspergillus niger genetically engineered bacteria is reduced and the yield of a biological membrane is reduced in the process of producing the citric acid through immobilized fermentation, the carrier agglomeration phenomenon is reduced, the problem of low oxygen and mass transfer efficiency caused by carrier agglomeration is solved, the citric acid production efficiency is improved, the continuous fermentation performance of the strains is enhanced, and the method is suitable for industrial production.

Specifically, a recombinant Aspergillus niger genetically engineered bacterium plate is scraped by a spore scraping liquid to prepare a spore liquid, and then the spore liquid is inoculated into a fermentation culture medium containing an immobilized carrier to prepare the citric acid through fermentation.

Wherein the spore liquid is inoculated in the fermentation culture medium according to the volume ratio of 0.1-1%, and the preferred volume ratio is 0.4%.

Wherein the immobilization medium for immobilization fermentation is polyurethane.

Wherein the content of the immobilized medium is 0.1-5 g/L fermentation medium, preferably 2 g/L fermentation medium.

Preferably, the immobilization medium of the immobilization fermentation is pretreated sequentially with NaOH and HCl.

Further preferably, the immobilization medium is soaked in NaOH, washed, soaked in HCl, washed and dried to obtain the product.

Wherein the concentration of the NaOH water is 0.5-2M, and is preferably 1M.

Wherein the soaking time in NaOH is 30 min-2 h, preferably 1 h.

Among them, the washing is preferably performed to be neutral.

Wherein the HCl concentration is 0.5-2M, preferably 1M; the solvent is water.

Wherein the soaking time in HCl is 30 min-2 h, preferably 1 h.

Wherein the immobilization medium is cubic, and preferably has a volume of 0.5 cm ³ A cube of (a).

Wherein the fermentation medium is prepared by enzymolysis of corn flour with liquefying enzyme, or by bran leachate.

The preparation method of the fermentation medium obtained by carrying out enzymolysis on the corn flour by using the liquefying enzyme comprises the following steps:

(i) Adding liquefying enzyme into the corn flour solution, and preserving heat to obtain a corn flour solution;

(ii) Adding a liquefying enzyme into the corn flour liquid obtained in the step (i), performing enzymolysis, and filtering to obtain a corn flour clear liquid;

(iii) And (3) mixing the corn flour liquid before filtration in the step (ii) with the corn flour clear liquid obtained in the step (ii).

In the step (i), the concentration of the corn flour solution is 200-300 g/L.

In step (i), the solvent of the corn flour solution is water.

In the steps (i) and (ii), the liquefying enzyme contains alpha-amylase, and the enzyme activity of the alpha-amylase is 70000-80000U/mL (the enzyme amount required by 1 min hydrolysis of 1 mg soluble starch is 1 enzyme activity unit, U/mL, under the conditions that the pH of the liquefying enzyme of 1 mL is 5.5 and the temperature is 85 ℃).

In the step (i), the dosage of the liquefying enzyme is 0.5-1 mL/L corn flour solution.

In step (i), the temperature of the heat preservation is 60-75 ℃.

In the step (i), the heat preservation time is 35-45 min.

In the step (ii), the corn flour liquid is heated to 85-105 ℃, and then the liquefying enzyme is added.

In the step (ii), the dosage of the liquefying enzyme is 0.5-1 mL/L corn flour liquid.

In the step (ii), the enzymolysis time is enzymolysis until the iodine solution does not turn blue, and is preferably 45-60 min.

In the step (iii), the volume ratio of the corn flour liquid to the corn flour clear liquid is 2-10:100.

wherein the concentration of each component in the fermentation medium prepared by the bran leachate is as follows: glucose 250 g/L, naNO ₃ 4 .2 g/L、KH ₂ PO ₄ 1.0g/L、MgSO ₄ ·7H ₂ O0.5 g/L, solvent is bran extract.

The preparation method of the bran leachate comprises the steps of boiling and filtering bran, preferably, boiling 1 h to obtain a bran water solution, filtering to obtain a filtrate, and fixing the volume by using water.

Wherein the fermentation temperature is 30-37 ℃.

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

Wherein the fermentation time is 72-115 h.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the invention provides an AcuM gene inactivated recombinant Aspergillus niger genetically engineered bacterium, which reduces the amount of a biological membrane of Aspergillus niger in the process of producing citric acid by immobilized fermentation, effectively relieves the phenomenon that the pore diameter of a carrier is blocked, and increases the oxygen and mass transfer effects of the carrier and the outside, thereby exerting the advantages of immobilized fermentation, improving the yield and the sugar-acid conversion rate of citric acid, shortening the fermentation period, and effectively solving the problems of low yield and long fermentation period in the prior industrial production of citric acid by Aspergillus niger fermentation; and the obtained recombinant Aspergillus niger genetically engineered bacteria can be fermented continuously in 12 batches, and the fermentation intensity is not obviously reduced.

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.

FIG. 2 is a PAN7-1 plasmid map.

FIG. 3 shows PCR electrophoresis of upstream and downstream homology arms of AcuM gene, wherein M is DNAmarker of DL 5000, lane 1 is the upper homology arm of AcuM gene, lane 2 is hyg resistance expression element, and lane 3 is the lower homology arm of AcuM gene.

FIG. 4 shows an electrophoretogram of knockout fragments, where M is marker lane and lane 1 is knockout fragment.

FIG. 5 is a graph showing crystal violet staining, wherein A is original bacteria and B is a.DELTA.AcuM strain.

FIG. 6 is a graph showing differences in OD values of crystal violet stains.

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

FIG. 8 is an electron micrograph of biofilm from pristine A.niger.

FIG. 9 is the electron microscope image of the biological membrane of the recombinant Aspergillus niger genetically engineered bacterium (delta AcuM strain).

Fig. 10 is a graph of the difference in production intensity of the Δ AcuM strain and the original strain after 12 batches 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 Aspergillus niger iron acquisition regulatory gene AcuM knock-out bacterium

Extraction of original Aspergillus niger genome

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

1. inoculating scraped Aspergillus Niger ATCC12846 spore liquid 1 mL into 50mL DP culture medium, culturing at 35 deg.C and 200 r/min for 24 h; the DP medium formulation is as follows: 10 g/L dextrin, 5g/L peptone, 2.5 g/L monopotassium phosphate, 1 g/L sodium nitrate, 0.5g/L magnesium sulfate, 10 g/L glycine, and the volume is increased to 100 mL by using water, and then the mixture is subpackaged into 50 mL.

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

3. Add 62.5. Mu.L Buffer KAC to step 2 and mix well. The mixture was kept on ice for 5min and centrifuged at 12000 rpm for 5min. The supernatant was collected at 600. Mu.L, added to 600. Mu.L of Buffer GB, and mixed well.

4. The Spin Column was mounted on a Collection Tube, the solution was transferred to the Spin Column, centrifuged at 12000 rpm for 1 min, and the filtrate was discarded.

5. mu.L of Buffer WA WAs added to Spin Column, centrifuged at 12000 rpm for 1 min, and the filtrate WAs discarded.

6. mu.L of Buffer WB was added to Spin Column, centrifuged at 12000 rpm for 1 min, and the filtrate was discarded.

7. Repeat step 6 once.

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

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

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

Amplifying an upstream homology arm by taking the original Aspergillus niger genome extracted in the step (one) as a template and AcuM-up-F and AcuM-up-R as an upstream primer and a downstream primer, wherein nucleotide sequences of the upstream homology arm and the downstream primer are respectively shown as SEQ ID No.3 and SEQ ID No. 4; acuM-down-F and AcuM-down-R are used as an upstream primer and a downstream primer to amplify downstream homologous arms, and the nucleotide sequences are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6.

The amplification reaction system is shown in Table 1, and the PCR reaction conditions are as follows: (1) denaturation at 95 ℃ of 10 s; (2) annealing at 55 ℃ for 30 s; (3) extension at 72 ℃ for 5 min; repeating the steps (1) to (3) for 35 times.

TABLE 1 PCR reaction System and reaction conditions

After the reaction, the PCR product was quantified by agarose gel electrophoresis, as shown in FIG. 3, lane 1 is the upper homology arm of AcuM gene, lane 3 is the lower homology arm of AcuM gene, the nucleotide sequence of the upstream homology arm of AcuM gene is shown in SEQ ID No.11, and the nucleotide sequence of the downstream homology arm of AcuM gene is shown in SEQ ID No. 12.

(III) amplification of hyg-resistant expression elements

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) is used as a template, acuM-hyg-F and AcuM-hyg-R are used as an upstream primer and a downstream primer to amplify hyg resistance expression elements, and the nucleotide sequences are respectively shown in SEQ ID NO.7 and SEQ ID NO. 8.

The reaction system is shown in Table 1, and the PCR reaction conditions are as follows: (1) denaturation at 5 ℃ of 10 s; (2) annealing at 55 ℃ for 30 s; (3) extension at 72 ℃ for 5 min; repeating the steps (1) to (3) for 35 times.

After the reaction, the PCR product was quantified by agarose gel electrophoresis, as shown in FIG. 3, lane 2 shows the hyg expression element, the nucleotide sequence of which is shown in SEQ ID NO. 13.

(IV) amplification of Gene knockout fragment

AcuM upstream homology arm, downstream homology arm and hyg resistance expression element are used as templates, acuM-F, acuM-R is used as an upstream primer and a downstream primer, respectively, and an overlapping extension PCR (Overlap PCR) technology is used for amplifying AcuM gene knockout fragments, wherein the nucleotide sequences of the AcuM gene knockout fragments are respectively shown as SEQ ID No.9 and SEQ ID No. 10.

The reaction system is shown in Table 1, and the PCR reaction conditions are as follows: (1) denaturation at 95 ℃ of 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, lane 1 shows the knockout fragment, and the nucleotide sequence of the knockout fragment is shown in 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, 3 mL scrape buffer was added to the plates, the spores were scraped off with a spreading stick and transferred to a sterilized 5 mL centrifuge tube.

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

2. Taking 0.5 mL obtained in step 1, inoculating the spore liquid into 50mL DP culture medium, culturing at 35 ℃,200 rpm for 13-16 h, performing microscopic examination, and observing the spore germination state.

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

Enzymatic hydrolysates (lyase Lysing enzyme, crashease, snailase, 0.1 g/10 mL, cellulase 400 μ L/10 mL, respectively) were prepared and sterilized with sterile syringe filter.

TABLE 2 manufacturer model of the enzymes used

4. Adding the hypha obtained in the step 3 of 2 g into 20 mL of the enzymolysis liquid obtained in the step 3, carrying out enzymolysis at 30 ℃ and 220 rpm for 30 min; then the rotating speed is reduced to 150 rpm for enzymolysis of 3 h.

5. After the enzymolysis is finished, filtering with filter paper, taking the filtrate, and centrifuging at 5000 rpm for 10 min at 4 ℃. Removing supernatant, adding 1 mL of 1M sorbitol aqueous solution (ice water bath), uniformly mixing by blowing and sucking with a gun, adding 15 mL of 1M sorbitol aqueous solution, centrifuging, and removing supernatant. And then repeated again. Removing the supernatant, adding 1 mL of Solution5, and uniformly mixing by blowing and sucking with a gun to obtain the protoplast. Wherein, the Solution5 is self-made, and the formula is as follows: KCl 4.47 g, caCl ₂ 0.735 g, MOP 0.2093 g, adjusting pH to 6.0 with KOH, and adding water to 100 mL.

6. And (3) sucking 100 mu L of protoplast into a 1.5 mL sterilized centrifugal tube, and adding 10 mu L of the gene knockout fragment constructed in the step (IV) to be uniformly mixed with the protoplast. Then adding 50 mu L of Solution4, mixing uniformly, placing on ice and timing for 15-30 min. Wherein, solution4 is self-made, and the formula is as follows: PEG8000 g, caCl ₂ 1.47 g, KCl 4.47 g,10 mM Tris, pH 7.5 adjusted with hydrochloric acid and made up to 100 mL with water.

7. Adding 900 μ L Solution4 after 20 min, turning upside down for several times, mixing, standing at room temperature and timing for 15-30 min. And (3) after 15-30 min, centrifuging at 6000 rpm for 5min, discarding 900 mu L of supernatant, coating the residual thallus in a PDA culture medium with sucrose concentration of 1 mol/L and hygromycin concentration of 150 mmol/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 transformant was added to 50. Mu.L of colony PCR buffer (100 mmol/L Tris-HCl, 10 mmol/L EDTA, 1 mol/L KCl), water-bathed at 95 ℃ for 10 min, and 0.5. Mu.L of transformant was added to the PCR reaction system. 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 the recombinant Aspergillus niger genetically engineered bacteria are obtained: a. AcuM strain.

Example 2: crystal violet staining test

Respectively inoculating original Aspergillus Niger ATCC12846 (original bacteria) and recombinant Aspergillus Niger genetically engineered bacteria (delta AcuM strains) to a PDA (personal digital assistant) plate, adding 3 mL spore scraping buffer solution into the PDA plate when spores are overgrown, scraping the spores by using a coating rod, transferring the spores into a sterilized 5 mL centrifugal tube, and fixing the volume to 2 mL by using the spore scraping buffer solution to obtain spore liquid. Quantitated and diluted to 10 using a hemocytometer ⁵ one/mL, followed by further dilution to 10 ⁴ /mL，10 ³ /mL。

A24-well plate was previously loaded with 1 mL synthetic medium, and then 2. Mu.L of spore solutions of different concentrations were inoculated into the medium. Standing and culturing at 35 ℃ for 36 h to enable aspergillus niger to form a film at the bottom of a 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 30 min to decolorize the crystal violet. Then, observation and detection of OD570 by microplate reader were carried out. FIG. 5 and FIG. 6 show the difference between the crystal violet staining pattern and the OD value.

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

TABLE 3 OD values of biofilm crystal violet staining experiments at different spore concentrations

The results in FIGS. 5 and 6 show that the.DELTA.AcuM strain, after destaining, is markedly lighter in purple than the original strain, at 10 ³ The color of the delta AcuM 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. Indicating that the biofilm is reduced after inactivation of the iron acquisition regulatory gene Δ AcuM.

Example 3: immobilized fermentation experiment of genetically engineered bacteria

1. Preparation of porous fiber material immobilized medium

Soaking polyurethane material in 1M NaOH aqueous solution to 1 h, washing with pure water, soaking in 1M HCl aqueous solution to 1 h, washing with pure water until pH is neutral, oven drying in 65 deg.C oven to constant weight, and cutting into 0.5 cm ³ The vectors with the same size are arranged on the left and the right.

2. Preparation of immobilized fermentation Medium

(i) Weighing 250 g/L corn flour, placing in a 75 ℃ water bath, adding 1 mL liquefying enzyme (60000U/L) when the corn flour liquid reaches 65 ℃, and preserving heat for 40 min to obtain the corn flour liquid;

(ii) Heating the water bath kettle to 95 ℃, adding 1 mL of liquefying enzyme when the corn flour liquid reaches 85 ℃, performing enzymolysis for 60 min until the iodine solution does not turn blue, and filtering to obtain a clear corn flour liquid;

(iii) Adding the unfiltered corn flour liquid obtained in the step (ii) as a feed back (the volume ratio of the unfiltered corn flour liquid to the corn flour clear liquid is 8 percent) into the corn flour clear liquid obtained in the step (ii), uniformly mixing, and subpackaging every 100 mL into 500 mL conical flasks containing 2 g/L carriers. Sterilizing and cooling for later use.

3. Fermentation of

(1) Inoculating frozen recombinant Aspergillus Niger genetically engineered bacteria (delta AcuM strain) and original Aspergillus Niger ATCC12846 (original bacteria) spores onto a PDA plate, culturing at 35 ℃ in a constant temperature incubator for 4-5 days, and allowing the spores to grow thereon.

(2) The spores are scraped by a spore scraping buffer solution to obtain a spore suspension, a proper amount of the spore suspension is taken and transferred into a 500 mL conical flask filled with 100 mL immobilized fermentation medium, the spore suspension is cultured in a shaking table at 35 ℃ and 250 rpm for 96 h, a sample is taken every 12 h in the fermentation process, the supernatant is centrifuged at 12000 rpm for 5min, and the residual sugar concentration and the citric acid yield in the supernatant are determined. When the residual sugar concentration is lower than 5g/L, the fermentation is finished. Wherein, the citric acid is measured by NaOH titration method, and the total sugar is measured by DNS method.

The NaOH titration method comprises the following steps: a sample of 1 mL (diluted to a certain concentration) was added to a 250mL Erlenmeyer flask, along with 50mL purified water, and titrated with 0.1429M NaOH, and the amount of NaOH consumed was the yield of citric acid.

The DNS method comprises the following steps: and (3) DNS preparation: 3,5-dinitrosalicylic acid 10 g is weighed, placed in about 60 mL of water, sodium hydroxide 10 g is gradually added, dissolved in a water bath at 50 ℃ by magnetic stirring, sodium methyl tartrate 200 g, phenol 2 g and anhydrous sodium sulfite 5g are sequentially added, after all the materials are dissolved and clarified, the solution is cooled to the room temperature, and pure water is used for fixing the volume to 1000 mL. Stored in a brown reagent bottle, and then placed in a dark place to use 7 d.

The preparation method of the standard yeast comprises the following steps: preparing a series of sugar standard solutions with the concentration of 0.0-1.0 g/L, respectively taking centrifuge tubes of 0.5 mL and 15 mL for each concentration, then adding 0.5 mL 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 8 mL pure water into each centrifuge tube, uniformly mixing, measuring the light absorption value OD540 under the wavelength of 540 nm, 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, 1 mL concentrated sulfuric acid is added into a 10 mL sample, the mixture is placed into ice water for cooling after reacting for 15 min in a boiling water bath, the pH is adjusted to be neutral, the volume is determined to be 100 mL, and then the mixture is diluted to the proper concentration, and the measurement method is the same as the measurement method. Different sugars correspond to different standard yeasts.

FIG. 7 shows the differences between the Δ AcuM strain and the original strain under fermentation conditions, and the results show that after fermentation at 96 h, the average yield of citric acid produced by fermentation of the Δ AcuM strain is 185.3 g/L, and the average yield of citric acid produced by fermentation of the original strain is 161.2 g/L. In the sugar-acid conversion rate, the conversion rate of the delta AcuM strain reaches 102.9 percent, is improved by 13.4 percent compared with that of the original strain, and the period is also reduced from 84 h to 72 h. Therefore, aspergillus Niger (ATCC 12846) is a high-yield citric acid strain, the yield of the delta AcuM strain can be improved on the basis, the period is obviously shortened, the continuous fermentation effect is obviously enhanced, so that the method is greatly improved on the basis of producing citric acid by immobilized fermentation of Aspergillus Niger and is suitable for industrial production.

(3) The immobilized fermented 4 d vector was taken out and washed 3 times with PBS to remove adsorbed mycelia. The amount of the original strain was excessive (FIG. 8) and the amount of the. DELTA. AcuM strain (FIG. 9) was moderate, thereby obtaining a carrier to which hyphae had adhered.

(4) And drying the mycelium-attached carrier, weighing, and subtracting the weight of the original carrier to obtain the weight of the attached membrane. The membrane content of the original strain is 5.34 g/L, the membrane content of the delta AcuM strain is 1.21 g/L, and the membrane content is reduced by 77.3%.

Example 4: immobilized continuous fermentation experiment of genetically engineered bacteria

Immobilized continuous batch fermentation was performed based on example 3, i.e. in step 3 (2), after each fermentation, the fermentation broth obtained from the fermentation was replaced with fresh immobilized fermentation medium, the fermentation was continued, and 12 batches were repeated.

Fig. 10 shows the difference in production intensity of the Δ AcuM strain and the original strain after 12 consecutive fermentations. As can be seen from the figure, the fermentation intensity of the AcuM strain is not obviously reduced after 12 batches of continuous fermentation, while the fermentation intensity of the original strain 12 batches is reduced by 69.1 percent, which shows that the continuous fermentation performance of the AcuM strain is obviously better than that of the original strain.

Example 5

Immobilized fermentation was performed on the basis of example 3, with the following modifications of the fermentation medium: glucose 250 g/L, naNO ₃ 4.2 g/L、KH ₂ PO ₄ 1.0 g/L、MgSO ₄ ·7H ₂ O0.5 g/L, prepared by bran leaching liquid; the preparation method of the bran leachate comprises the following steps: weighing bran 15 g, adding into bran 800Boiling 1 h with mL water, filtering to obtain filtrate, and adding water to volume of 1L.

The conditions of the immobilized fermentation were as follows: 72 h was cultured at 220 r/min at 30 ℃.

After 72 h is fermented and cultured, the yield of the delta AcuM strain citric acid reaches 261.1 g/L, and the sugar-acid conversion rate reaches 104.4%.

The invention provides a recombinant aspergillus niger genetically engineered bacterium, a construction method and an application concept and a method, and a plurality of methods and ways for realizing the technical scheme are provided, the above description is only a preferred embodiment of the invention, and it should be noted that, for a person skilled in the art, a plurality of improvements and decorations can be made without departing from the principle of the invention, and the improvements and decorations should be regarded as the protection scope of the invention. All the components not specified in this embodiment can be implemented by the prior art.

Sequence listing

<110> Nanjing university of industry

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

<160> 17

<170> SIPOSequenceListing 1.0

<210> 1

<211> 6079

<212> DNA

<213> Aspergillus niger (Aspergillus niger)

<400> 1

aaggaccatc aaatccaagt ggtccacact tccggagcct gttcaagaca gggtgcgcga 60

catgtttcgg gccctggagc gtccggttat cgtaagacag cagaacgagg gcaaacgtat 120

cgaagcacag gctgctgttc aggctgttgt gaaaaagtat agttctttgc cttttggctc 180

gaggagaatg atcatactaa tttgtatttc ccaactgtat agtctaggaa aaaggcttcc 240

gcgcatgcca tttcctccag ttacgaaaga ctctgtcttt gagtacgagg cagcgctcaa 300

ggaacatgta agtaatgctc aattaaacct ctttgccaca aggtataact gacatatttt 360

gcagtgctcc ttggaagcta ctttagccac agtgactgat agtattgatc tcttaaaggc 420

tgaagtcgaa aaggaagaag ctatgctcga aagagagaaa aagcaacttc aggagatgga 480

gaggaatgcg aagcgggcgg aagcggagag gaagaggcaa tcaaagaacg taagtgtgac 540

cttgatgcga ataactcatg aattttacgc tgatcttttc taggaacatc ctgttctgcg 600

gcaggtcgac agcttgcctg atgagtataa tcagaagcag accgagttta cccttgcggg 660

aggaaaagaa acccagacga tgttcgacga ggtgcgtttt cagctcccct tgtaccttcg 720

cctgatgtcc gttgctgaca taccgtgttt agcttgaaaa tgacccagaa gtctcgaata 780

tcttgaaaca actgactgga catctgaagt caatgcaaaa caacaccgca cccgtcgctg 840

gtttgaagga cgcaattggc aggtcacatg cagcattaag tctgactttc atgcccgaag 900

actgaactta cagaattctg catcaatggc aagttcagaa cgagtgtgct gttacagcgt 960

gtacgacatg tggtgcgtat atcgaaagat gcaagcccat gccagtcttt aagacagctt 1020

ggaaaacgag tccatgcgta ggacggtatt cgacatcatg caatggacca aggtacctgc 1080

gagccagttt tgacgctacc tggaatggct atcgtctcaa ctgtgtgcaa aacccctggc 1140

gggacaccag tgctatacgg ggctaaacga attcccgcgc cttggagacc tagtcctcct 1200

attcaggaca agctaagccc tcatagcgtc ctaccacgtt ctcgtcagga attaggctct 1260

gggaattctt taggttcaag tggagtttag ccaaatattg acagcggacc cccaccagtt 1320

gactataggg aaagatgtcc agtgggctta gaggaaggtc gtgtctgggg atgctaatga 1380

ggcatcatct acgggttttt tgccacccta aaaccactta atacaacacc taattctgaa 1440

gaggaaacaa attactccat tgcctactct acagaacagc ttaatgtagg aacatgtggc 1500

tgcatatgct aggagcgagt taaccgaggt agaacccttg tagctatttc ccataacaga 1560

tagtcgttct atataccaac atcagaaata cgaggatcct gttgattgtg tatctacgtg 1620

atcgcgactc aaacaattga tattggctgc tttgctttct gcagtgagga tagtgttgca 1680

catgagttag ccgctcatat gcttttccga ggctactaac cggttcgccg ctgccggccc 1740

caatgcggag gggcccggga aatgaaagat ccaagtcccg caccagcggg gaacttcgat 1800

gagccgatcg catcgcgcgt gccctcacat tttcacgtta cagatcagcc cgatcccctc 1860

acccttctgc ttaatcggtc cattcaagcc cagtcgaatg tctgcagacg gctattttgc 1920

tccccgtttg tgttccgatc gacagcaaca gtggtaacca cgaaccgaac cctgcgcaac 1980

acctttgcgc ctagcgatca atgacggaga aaactgcggc gtcaacgggt ccaggccccg 2040

atcaacaaac cagcaatggg acagcagata gaacaaaacc ggatgtgacg gaggaggagc 2100

agactggggc agatgcgagt gcgaaagaca actcgcagaa tacaccgtcc ctcaaggcgg 2160

atggtgccgc cgcgggggct cactcaagtc ctaagaaacg tcgcaaagtc aatcatggta 2220

tggcgctatt tgctatgcat cctacaagga tccaggcagc gacttgcatt tttaccttgt 2280

tgtttcgacc acgatgctaa ccagagtgcc acgtttgtag catgcgttta ttgccgacga 2340

tcggtaaagt ctttcccctc aaacaagatt accttcctcg cccctcactt cagtcaacac 2400

gagccgacat cgtccttgat agttgttggg ctcaaggtat agaggagtac tttgctaact 2460

gaaagacact cgctcgcgtg gcacagcata tgacttgcga ctcggtacga cccgtctgag 2520

ctctaccctc cagcgcattc ccgtggaaca tggagtgcaa tcacttttcg agattccagt 2580

tgctgacggt tgttgctttg cgggaaatgt aggagcgacc ctgcacgcga tgcataaaac 2640

ggaatattgg ccacctttgc cacgatgaac ctcgggaacc ctcaaaacgg tcacggagcg 2700

aaaacgatca ttcggccgct gatgacgagg gctcttctaa taatgagtac tccggcgttc 2760

aaatgcccag aaacgtcgac ggcccagatg ctgcaggcca gcaaatcata cccgacggga 2820

ccatcggcct acccacctct tctgtgactt ctgtgcagca gcctggcaac atggcttctt 2880

ctggccaggg tcttaatgct caacagcaaa gtgagtgcca cccttataaa attgtccagt 2940

gtatcataat aacattgggt agtgattggg tacaacgaat ggattggtgg acaaagccag 3000

tttcaggaca tgcacacgtt tcacccttct tacatgttca atgcaccaga ggtcactaat 3060

gagtacaacc ttcttggtga ctttttgagc aatagcctct tggatgatgg aggagtattc 3120

cagaacgatg aactgcaggg aatctactcg gacccgtcgt tactcaattc catggcgact 3180

ctgggaaatt cgaatcaaag ccttcttcag cagtcccaac ctgcgcaacc tcaaccgaat 3240

caacaggccc agggcgagcc tttacaaggg cctacgcctg cagtgagcaa tgataaggcg 3300

agagagacct attacatgac cgcagcagat ccatcagggt ccgacccgcc agaggagcgg 3360

atgaacaagc ttctcaaagc gaaatacgac gcgggacttc ttcgcccatt taactacgtc 3420

aagggttatg ccaggctgaa tcaatatatg gagcgacact tgcaacaagc ctcgcggcag 3480

aagatcctcc gacaattaga taaattccgg ccaaagttcc gggagcgaat gcagagcctg 3540

accgacattg agctcatcct cgtggagatg tggttcgaaa gaagcttgat ggaatatgat 3600

cgagtttttg ctagtatggc cattcctgcc tgttgctgga gacgaacggg tgaaatattc 3660

cgtggcaaca aagaaatggc agagttgatc ggcgtaccaa tcgagactct ccgagacgta 3720

agtgcattct gcagaaatct ctatgacaaa aatctaatat gactgtcata ctcagggaaa 3780

gcttgcaatc cacgagatca tcgtggaaga tcagctagtg agctattggg agaaattcgg 3840

agctattgct tttgataaca cgcaaaaagc catgctcaca agctgcaccc taaagaatcc 3900

caactccaac tcccccggag atgggatccc ctgttgcttc tcgtttacta ttcgaagaga 3960

tacccataac atgtgagtga gctcatcaga tgacgcggtg gcaaggtgct aactgctgtt 4020

gatatagccc atcactcatt attgggaatt tcctgccgtc acagcgaaag gccaaataag 4080

ccctggagtt tttttttatc attttatttc tctatcattg cccagcaaat gttcgcgtgg 4140

gctgatgcct ggttactgtc ttctttcttg gtgcttactg gcccttctga tggaggagcc 4200

tcgcgatatt gtttccttca tacccttctg tcattgattt atacggcctt caaaaatgta 4260

ccataacatc ttcaaggcgt gactcgtttc caagggaagg ttaattgtgg tcttgcaacg 4320

cgaaccgaaa atggactaga taggtcaggt gtcatctcgt acttcgcttc gggtgtcgac 4380

cgagtatccg tagcactaca ttcctcatct ttaaagccat taggacaacg actagagtta 4440

accacaagcg ctgctattga catgacacat cacagctcaa aagcacacat ggacgtcgtt 4500

ctaggtgcag cacctttccc gcatgccgtt tcatacctct cattgcctca tctactaaga 4560

acgaagcaac agacttcgca gctgagctaa ccaatagtct cgttatcgac caaaagaagc 4620

ctcatcttgg tctagatgat aaaatcttgg ctcaactgga tcggcaattc aagcactttc 4680

ctctgctgcc agcgagtccc tcgtctccga ctcgtcgcgg gtttgaccaa gaaggaacaa 4740

agctatggaa tatttgcatg caactcatga ctgtctatag agacagaagt gaagaccttc 4800

tccttgcgtg caaaggtact actgtgggat cccaaggaca cagactttcg ctgagtacag 4860

tatagtgaag gcattcgctt acgcgatgtt ggactatgcc gcaccatatc aaggccaggg 4920

taagtataac acacttatct attgtaagga ttcgagaaag ctaatattac caggaagcaa 4980

cagggcctta gaggcagcct tcagcatagc aatgacgtgc ataggtaaga gcagatcctt 5040

gttgctttct ccaatttccc ttgcacgtag ctctaacatc ttgcatatag acaacgactg 5100

tctcagtcta agccagaaaa tcatcgaagt cgcagctgtg cggttagata aacttgagag 5160

atatgaaagc gatgtcgaaa attctaagct tcaacaatat accatcgagt actacatgat 5220

tcgtgcccac ttggtatggt ctacccgtct gtaataatac acatcagact gacagctggg 5280

acctaagtag gcctggctgc agggaagact cgacatcgca gaacacttgt tctcgaagat 5340

accagtatct gataatggca ggggtcaaga acgtgtcatg gacatatgct ataagatcgg 5400

taactgtgcg atttcacaca agcagtatga tgtctctatg aagtggctag aaagagcgtt 5460

gagggcatgt gagtccatta ggcatatgca acaggcatcg atactgtcaa acaaggataa 5520

agagctcttg atacttcatg cttctggtag ggtgctcctg gttgatgaca aagttaagtt 5580

tgttcacaaa gaatagttcg tgccggcctt catcttgatc ctgaagagca cagtggcttc 5640

ctttctgaag ctctggatgc attgaaattc gtaagtcttg atatccagtt gttatatatt 5700

gctaatattt aacagcgtta cggtggcatg ttccctgtcc aggtcatcca acttgagatg 5760

ctggacaagg aaggagcaga tgagattgtc ttttcgcaag gtgactgaat tatagttcgt 5820

gattgttttc cctcttcgac tgacataaga gttaacagtg ctacaaagta ccattgaatc 5880

gcctgagctt aaagactcac acctcgcaat gtaatggcca atatgaactc gtgtacgccg 5940

tccggtacta acaaagtggg tgtagcatca tgtattatat acagaagctg tttaatacat 6000

cgtgagcgca ccggtcacat ctgtctgctc atctgacaag taaccagtcc cgaggtctcc 6060

atcagtatgc tcaagcaac 6079

<210> 2

<211> 6740

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

aaggaccatc aaatccaagt ggtccacact tccggagcct gttcaagaca gggtgcgcga 60

catgtttcgg gccctggagc gtccggttat cgtaagacag cagaacgagg gcaaacgtat 120

cgaagcacag gctgctgttc aggctgttgt gaaaaagtat agttctttgc cttttggctc 180

gaggagaatg atcatactaa tttgtatttc ccaactgtat agtctaggaa aaaggcttcc 240

gcgcatgcca tttcctccag ttacgaaaga ctctgtcttt gagtacgagg cagcgctcaa 300

ggaacatgta agtaatgctc aattaaacct ctttgccaca aggtataact gacatatttt 360

gcagtgctcc ttggaagcta ctttagccac agtgactgat agtattgatc tcttaaaggc 420

tgaagtcgaa aaggaagaag ctatgctcga aagagagaaa aagcaacttc aggagatgga 480

gaggaatgcg aagcgggcgg aagcggagag gaagaggcaa tcaaagaacg taagtgtgac 540

cttgatgcga ataactcatg aattttacgc tgatcttttc taggaacatc ctgttctgcg 600

gcaggtcgac agcttgcctg atgagtataa tcagaagcag accgagttta cccttgcggg 660

aggaaaagaa acccagacga tgttcgacga ggtgcgtttt cagctcccct tgtaccttcg 720

cctgatgtcc gttgctgaca taccgtgttt agcttgaaaa tgacccagaa gtctcgaata 780

tcttgaaaca actgactgga catctgaagt caatgcaaaa caacaccgca cccgtcgctg 840

gtttgaagga cgcaattggc aggtcacatg cagcattaag tctgactttc atgcccgaag 900

actgaactta cagaattctg catcaatggc aagttcagaa cgagtgtgct gttacagcgt 960

gtacgacatg tggtgcgtat atcgaaagat gcaagcccat gccagtcttt aagacagctt 1020

ggaaaacgag tccatgcgta ggacggtatt cgacatcatg caatggacca aggtacctgc 1080

gagccagttt tgacgctacc tggaatggct atcgtctcaa ctgtgtgcaa aacccctggc 1140

gggacaccag tgctatacgg ggctaaacga attcccgcgc cttggagacc tagtcctcct 1200

attcaggaca agctaagccc tcatagcgtc ctaccacgtt ctcgtcagga attaggctct 1260

gggaattctt taggttcaag tggagtttag ccaaatattg acagcggacc cccaccagtt 1320

gactataggg aaagatgtcc agtgggctta gaggaaggtc gtgtctgggg atgctaatga 1380

ggcatcatct acgggttttt tgccacccta aaaccactta atacaacacc taattctgaa 1440

gaggaaacaa attactccat tgcctactct acagaacagc ttaatgtagg aacatgtggc 1500

tgcatatgct aggagcgagt taaccgaggt agaacccttg tagctatttc ccataacaga 1560

tagtcgttct atataccaac atcagaaata cgaggatcct gttgattgtg tatctacgtg 1620

atcgcgactc aaacaattga tattggctgc tttgctttct gcagtgagga tagtgttgca 1680

catgagttag ccgctcatat gcttttccga ggctactaac cggttcgccg ctgccggccc 1740

caatgcggag gggcccggga aatgaaagat ccaagtcccg caccagcggg gaacttcgat 1800

gagccgatcg catcgcgcgt gccctcacat tttcacgtta cagatcagcc cgatcccctc 1860

acccttctgc ttaatcggtc cattcaagcc cagtcgaatg tctgcagacg gctattttgc 1920

tccccgtttg tgttccgatc gacagcaaca gtggtaacca cgaaccgaac cctgcgcaac 1980

acctttgcgc ctagcgatca taaaatccgc cgcctccacc atttgtagaa aaatgtgacg 2040

aactcgtgag ctctgtacag tgaccggtga ctctttctgg catgcggaga gacggacgga 2100

cgcagagaga agggctgagt aataagccac tggccagaca gctctggcgg ctctgaggtg 2160

cagtggatga ttattaatcc gggaccggcc gcccctccgc cccgaagtgg aaaggctggt 2220

gtgcccctcg ttgaccaaga atctattgca tcatcggaga atatggagct tcatcgaatc 2280

accggcagta agcgaaggag aatgtgaagc caggggtgta tagccgtcgg cgaaatagca 2340

tgccattaac ctaggtacag aagtccaatt gcttccgatc tggtaaaaga ttcacgagat 2400

agtaccttct ccgaagtagg tagagcgagt acccggcgcg taagctccct aattggccca 2460

tccggcatct gtagggcgtc caaatatcgt gcctctcctg ctttgcccgg tgtatgaaac 2520

cggaaaggcc gctcaggagc tggccagcgg cgcagaccgg gaacacaagc tggcagtcga 2580

cccatccggt gctctgcact cgacctgctg aggtccctca gtccctggta ggcagctttg 2640

ccccgtctgt ccgcccggtg tgtcggcggg gttgacaagg tcgttgcgtc agtccaacat 2700

ttgttgccat attttcctgc tctccccacc agctgctctt ttcttttctc tttcttttcc 2760

catcttcagt atattcatct tcccatccaa gaacctttat ttcccctaag taagtacttt 2820

gctacatcca tactccatcc ttcccatccc ttattccttt gaacctttca gttcgagctt 2880

tcccacttca tcgcagcttg actaacagct accccgcttg agcagacatc accatgcctg 2940

aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc 3000

tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg 3060

gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc 3120

ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg 3180

agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg 3240

aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg 3300

ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca 3360

ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg 3420

tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg 3480

ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc 3540

tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt 3600

cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc 3660

agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt 3720

atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg 3780

atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg 3840

ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac 3900

tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa tagagtagat 3960

gccgaccgcg ggatccactt aacgttactg aaatcatcaa acagcttgac gaatctggat 4020

ataagatcgt tggtgtcgat gtcagctccg gagttgagac aaatggtgtt caggatctcg 4080

ataagatacg ttcatttgtc caagcagcaa agagtgcctt ctagtgattt aatagctcca 4140

tgtcaacaag aataaaacgc gttttcgggt ttacctcttc cagatacagc tcatctgcaa 4200

tgcattaatg cattgactgc aacctagtaa cgccttncag gctccggcga agagaagaat 4260

agcttagcag agctattttc attttcggga gacgagatca agcagatcaa cggtcgtcaa 4320

gagacctacg agactgagga atccgctctt ggctccacgc gactatatat ttgtctctaa 4380

ttgtactttg acatgctcct cttctttact ctgatagctt gactatgaaa attccgtcac 4440

cagcncctgg gttcgcaaag ataattgcat gtttcttcct tgaactctca agcctacagg 4500

acacacattc atcgtaggta taaacctcga aatcanttcc tactaagatg gtatacaata 4560

gtaaccatgc atggttgcct agtgaatgct ccgtaacacc caatacgccg gccgaaactt 4620

ttttacaact ctcctatgag tcgtttaccc agaatgcaca ggtacacttg tttagaggta 4680

atccttcttt ctagaagtcc tcgtgtactg tgtaagcgcc cactccacat ctccactcga 4740

gccctggagt ttttttttat cattttattt ctctatcatt gcccagcaaa tgttcgcgtg 4800

ggctgatgcc tggttactgt cttctttctt ggtgcttact ggcccttctg atggaggagc 4860

ctcgcgatat tgtttccttc atacccttct gtcattgatt tatacggcct tcaaaaatgt 4920

accataacat cttcaaggcg tgactcgttt ccaagggaag gttaattgtg gtcttgcaac 4980

gcgaaccgaa aatggactag ataggtcagg tgtcatctcg tacttcgctt cgggtgtcga 5040

ccgagtatcc gtagcactac attcctcatc tttaaagcca ttaggacaac gactagagtt 5100

aaccacaagc gctgctattg acatgacaca tcacagctca aaagcacaca tggacgtcgt 5160

tctaggtgca gcacctttcc cgcatgccgt ttcatacctc tcattgcctc atctactaag 5220

aacgaagcaa cagacttcgc agctgagcta accaatagtc tcgttatcga ccaaaagaag 5280

cctcatcttg gtctagatga taaaatcttg gctcaactgg atcggcaatt caagcacttt 5340

cctctgctgc cagcgagtcc ctcgtctccg actcgtcgcg ggtttgacca agaaggaaca 5400

aagctatgga atatttgcat gcaactcatg actgtctata gagacagaag tgaagacctt 5460

ctccttgcgt gcaaaggtac tactgtggga tcccaaggac acagactttc gctgagtaca 5520

gtatagtgaa ggcattcgct tacgcgatgt tggactatgc cgcaccatat caaggccagg 5580

gtaagtataa cacacttatc tattgtaagg attcgagaaa gctaatatta ccaggaagca 5640

acagggcctt agaggcagcc ttcagcatag caatgacgtg cataggtaag agcagatcct 5700

tgttgctttc tccaatttcc cttgcacgta gctctaacat cttgcatata gacaacgact 5760

gtctcagtct aagccagaaa atcatcgaag tcgcagctgt gcggttagat aaacttgaga 5820

gatatgaaag cgatgtcgaa aattctaagc ttcaacaata taccatcgag tactacatga 5880

ttcgtgccca cttggtatgg tctacccgtc tgtaataata cacatcagac tgacagctgg 5940

gacctaagta ggcctggctg cagggaagac tcgacatcgc agaacacttg ttctcgaaga 6000

taccagtatc tgataatggc aggggtcaag aacgtgtcat ggacatatgc tataagatcg 6060

gtaactgtgc gatttcacac aagcagtatg atgtctctat gaagtggcta gaaagagcgt 6120

tgagggcatg tgagtccatt aggcatatgc aacaggcatc gatactgtca aacaaggata 6180

aagagctctt gatacttcat gcttctggta gggtgctcct ggttgatgac aaagttaagt 6240

ttgttcacaa agaatagttc gtgccggcct tcatcttgat cctgaagagc acagtggctt 6300

cctttctgaa gctctggatg cattgaaatt cgtaagtctt gatatccagt tgttatatat 6360

tgctaatatt taacagcgtt acggtggcat gttccctgtc caggtcatcc aacttgagat 6420

gctggacaag gaaggagcag atgagattgt cttttcgcaa ggtgactgaa ttatagttcg 6480

tgattgtttt ccctcttcga ctgacataag agttaacagt gctacaaagt accattgaat 6540

cgcctgagct taaagactca cacctcgcaa tgtaatggcc aatatgaact cgtgtacgcc 6600

gtccggtact aacaaagtgg gtgtagcatc atgtattata tacagaagct gtttaataca 6660

tcgtgagcgc accggtcaca tctgtctgct catctgacaa gtaaccagtc ccgaggtctc 6720

catcagtatg ctcaagcaac 6740

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aaggaccatc aaatccaagt 20

<210> 4

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ggtggaggcg gcggatttta tgatcgctag gcgcaaaggt 40

<210> 5

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cactccacat ctccactcga gccctggagt ttttttttat 40

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gttgcttgag catactgatg 20

<210> 7

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

acctttgcgc ctagcgatca taaaatccgc cgcctccacc 40

<210> 8

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ataaaaaaaa actccagggc tcgagtggag atgtggagtg 40

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aaggaccatc aaatccaagt 20

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gttgcttgag catactgatg 20

<210> 11

<211> 2000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

aaggaccatc aaatccaagt ggtccacact tccggagcct gttcaagaca gggtgcgcga 60

catgtttcgg gccctggagc gtccggttat cgtaagacag cagaacgagg gcaaacgtat 120

cgaagcacag gctgctgttc aggctgttgt gaaaaagtat agttctttgc cttttggctc 180

gaggagaatg atcatactaa tttgtatttc ccaactgtat agtctaggaa aaaggcttcc 240

gcgcatgcca tttcctccag ttacgaaaga ctctgtcttt gagtacgagg cagcgctcaa 300

ggaacatgta agtaatgctc aattaaacct ctttgccaca aggtataact gacatatttt 360

gcagtgctcc ttggaagcta ctttagccac agtgactgat agtattgatc tcttaaaggc 420

tgaagtcgaa aaggaagaag ctatgctcga aagagagaaa aagcaacttc aggagatgga 480

gaggaatgcg aagcgggcgg aagcggagag gaagaggcaa tcaaagaacg taagtgtgac 540

cttgatgcga ataactcatg aattttacgc tgatcttttc taggaacatc ctgttctgcg 600

gcaggtcgac agcttgcctg atgagtataa tcagaagcag accgagttta cccttgcggg 660

aggaaaagaa acccagacga tgttcgacga ggtgcgtttt cagctcccct tgtaccttcg 720

cctgatgtcc gttgctgaca taccgtgttt agcttgaaaa tgacccagaa gtctcgaata 780

tcttgaaaca actgactgga catctgaagt caatgcaaaa caacaccgca cccgtcgctg 840

gtttgaagga cgcaattggc aggtcacatg cagcattaag tctgactttc atgcccgaag 900

actgaactta cagaattctg catcaatggc aagttcagaa cgagtgtgct gttacagcgt 960

gtacgacatg tggtgcgtat atcgaaagat gcaagcccat gccagtcttt aagacagctt 1020

ggaaaacgag tccatgcgta ggacggtatt cgacatcatg caatggacca aggtacctgc 1080

gagccagttt tgacgctacc tggaatggct atcgtctcaa ctgtgtgcaa aacccctggc 1140

gggacaccag tgctatacgg ggctaaacga attcccgcgc cttggagacc tagtcctcct 1200

attcaggaca agctaagccc tcatagcgtc ctaccacgtt ctcgtcagga attaggctct 1260

gggaattctt taggttcaag tggagtttag ccaaatattg acagcggacc cccaccagtt 1320

gactataggg aaagatgtcc agtgggctta gaggaaggtc gtgtctgggg atgctaatga 1380

ggcatcatct acgggttttt tgccacccta aaaccactta atacaacacc taattctgaa 1440

gaggaaacaa attactccat tgcctactct acagaacagc ttaatgtagg aacatgtggc 1500

tgcatatgct aggagcgagt taaccgaggt agaacccttg tagctatttc ccataacaga 1560

tagtcgttct atataccaac atcagaaata cgaggatcct gttgattgtg tatctacgtg 1620

atcgcgactc aaacaattga tattggctgc tttgctttct gcagtgagga tagtgttgca 1680

catgagttag ccgctcatat gcttttccga ggctactaac cggttcgccg ctgccggccc 1740

caatgcggag gggcccggga aatgaaagat ccaagtcccg caccagcggg gaacttcgat 1800

gagccgatcg catcgcgcgt gccctcacat tttcacgtta cagatcagcc cgatcccctc 1860

acccttctgc ttaatcggtc cattcaagcc cagtcgaatg tctgcagacg gctattttgc 1920

tccccgtttg tgttccgatc gacagcaaca gtggtaacca cgaaccgaac cctgcgcaac 1980

acctttgcgc ctagcgatca 2000

<210> 12

<211> 2000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gccctggagt ttttttttat cattttattt ctctatcatt gcccagcaaa tgttcgcgtg 60

ggctgatgcc tggttactgt cttctttctt ggtgcttact ggcccttctg atggaggagc 120

ctcgcgatat tgtttccttc atacccttct gtcattgatt tatacggcct tcaaaaatgt 180

accataacat cttcaaggcg tgactcgttt ccaagggaag gttaattgtg gtcttgcaac 240

gcgaaccgaa aatggactag ataggtcagg tgtcatctcg tacttcgctt cgggtgtcga 300

ccgagtatcc gtagcactac attcctcatc tttaaagcca ttaggacaac gactagagtt 360

aaccacaagc gctgctattg acatgacaca tcacagctca aaagcacaca tggacgtcgt 420

tctaggtgca gcacctttcc cgcatgccgt ttcatacctc tcattgcctc atctactaag 480

aacgaagcaa cagacttcgc agctgagcta accaatagtc tcgttatcga ccaaaagaag 540

cctcatcttg gtctagatga taaaatcttg gctcaactgg atcggcaatt caagcacttt 600

cctctgctgc cagcgagtcc ctcgtctccg actcgtcgcg ggtttgacca agaaggaaca 660

aagctatgga atatttgcat gcaactcatg actgtctata gagacagaag tgaagacctt 720

ctccttgcgt gcaaaggtac tactgtggga tcccaaggac acagactttc gctgagtaca 780

gtatagtgaa ggcattcgct tacgcgatgt tggactatgc cgcaccatat caaggccagg 840

gtaagtataa cacacttatc tattgtaagg attcgagaaa gctaatatta ccaggaagca 900

acagggcctt agaggcagcc ttcagcatag caatgacgtg cataggtaag agcagatcct 960

tgttgctttc tccaatttcc cttgcacgta gctctaacat cttgcatata gacaacgact 1020

gtctcagtct aagccagaaa atcatcgaag tcgcagctgt gcggttagat aaacttgaga 1080

gatatgaaag cgatgtcgaa aattctaagc ttcaacaata taccatcgag tactacatga 1140

ttcgtgccca cttggtatgg tctacccgtc tgtaataata cacatcagac tgacagctgg 1200

gacctaagta ggcctggctg cagggaagac tcgacatcgc agaacacttg ttctcgaaga 1260

taccagtatc tgataatggc aggggtcaag aacgtgtcat ggacatatgc tataagatcg 1320

gtaactgtgc gatttcacac aagcagtatg atgtctctat gaagtggcta gaaagagcgt 1380

tgagggcatg tgagtccatt aggcatatgc aacaggcatc gatactgtca aacaaggata 1440

aagagctctt gatacttcat gcttctggta gggtgctcct ggttgatgac aaagttaagt 1500

ttgttcacaa agaatagttc gtgccggcct tcatcttgat cctgaagagc acagtggctt 1560

cctttctgaa gctctggatg cattgaaatt cgtaagtctt gatatccagt tgttatatat 1620

tgctaatatt taacagcgtt acggtggcat gttccctgtc caggtcatcc aacttgagat 1680

gctggacaag gaaggagcag atgagattgt cttttcgcaa ggtgactgaa ttatagttcg 1740

tgattgtttt ccctcttcga ctgacataag agttaacagt gctacaaagt accattgaat 1800

cgcctgagct taaagactca cacctcgcaa tgtaatggcc aatatgaact cgtgtacgcc 1860

gtccggtact aacaaagtgg gtgtagcatc atgtattata tacagaagct gtttaataca 1920

tcgtgagcgc accggtcaca tctgtctgct catctgacaa gtaaccagtc ccgaggtctc 1980

catcagtatg ctcaagcaac 2000

<210> 13

<211> 1192

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atcttcagta tattcatctt cccatccaag aacctttatt tcccctaagt aagtactttg 60

ctacatccat actccatcct tcccatccct tattcctttg aacctttcag ttcgagcttt 120

cccacttcat cgcagcttga ctaacagcta ccccgcttga gcagacatca ccatgcctga 180

actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct 240

gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg 300

atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg 360

gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aattcagcga 420

gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga 480

aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga tcgctgcggc 540

cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac 600

tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt 660

gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc 720

cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct 780

gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc 840

ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca 900

gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta 960

tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga 1020

tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg 1080

gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact 1140

cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat ag 1192

<210> 14

<211> 6740

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

aaggaccatc aaatccaagt ggtccacact tccggagcct gttcaagaca gggtgcgcga 60

catgtttcgg gccctggagc gtccggttat cgtaagacag cagaacgagg gcaaacgtat 120

cgaagcacag gctgctgttc aggctgttgt gaaaaagtat agttctttgc cttttggctc 180

gaggagaatg atcatactaa tttgtatttc ccaactgtat agtctaggaa aaaggcttcc 240

gcgcatgcca tttcctccag ttacgaaaga ctctgtcttt gagtacgagg cagcgctcaa 300

ggaacatgta agtaatgctc aattaaacct ctttgccaca aggtataact gacatatttt 360

gcagtgctcc ttggaagcta ctttagccac agtgactgat agtattgatc tcttaaaggc 420

tgaagtcgaa aaggaagaag ctatgctcga aagagagaaa aagcaacttc aggagatgga 480

gaggaatgcg aagcgggcgg aagcggagag gaagaggcaa tcaaagaacg taagtgtgac 540

cttgatgcga ataactcatg aattttacgc tgatcttttc taggaacatc ctgttctgcg 600

gcaggtcgac agcttgcctg atgagtataa tcagaagcag accgagttta cccttgcggg 660

aggaaaagaa acccagacga tgttcgacga ggtgcgtttt cagctcccct tgtaccttcg 720

cctgatgtcc gttgctgaca taccgtgttt agcttgaaaa tgacccagaa gtctcgaata 780

tcttgaaaca actgactgga catctgaagt caatgcaaaa caacaccgca cccgtcgctg 840

gtttgaagga cgcaattggc aggtcacatg cagcattaag tctgactttc atgcccgaag 900

actgaactta cagaattctg catcaatggc aagttcagaa cgagtgtgct gttacagcgt 960

gtacgacatg tggtgcgtat atcgaaagat gcaagcccat gccagtcttt aagacagctt 1020

ggaaaacgag tccatgcgta ggacggtatt cgacatcatg caatggacca aggtacctgc 1080

gagccagttt tgacgctacc tggaatggct atcgtctcaa ctgtgtgcaa aacccctggc 1140

gggacaccag tgctatacgg ggctaaacga attcccgcgc cttggagacc tagtcctcct 1200

attcaggaca agctaagccc tcatagcgtc ctaccacgtt ctcgtcagga attaggctct 1260

gggaattctt taggttcaag tggagtttag ccaaatattg acagcggacc cccaccagtt 1320

gactataggg aaagatgtcc agtgggctta gaggaaggtc gtgtctgggg atgctaatga 1380

ggcatcatct acgggttttt tgccacccta aaaccactta atacaacacc taattctgaa 1440

gaggaaacaa attactccat tgcctactct acagaacagc ttaatgtagg aacatgtggc 1500

tgcatatgct aggagcgagt taaccgaggt agaacccttg tagctatttc ccataacaga 1560

tagtcgttct atataccaac atcagaaata cgaggatcct gttgattgtg tatctacgtg 1620

atcgcgactc aaacaattga tattggctgc tttgctttct gcagtgagga tagtgttgca 1680

catgagttag ccgctcatat gcttttccga ggctactaac cggttcgccg ctgccggccc 1740

caatgcggag gggcccggga aatgaaagat ccaagtcccg caccagcggg gaacttcgat 1800

gagccgatcg catcgcgcgt gccctcacat tttcacgtta cagatcagcc cgatcccctc 1860

acccttctgc ttaatcggtc cattcaagcc cagtcgaatg tctgcagacg gctattttgc 1920

tccccgtttg tgttccgatc gacagcaaca gtggtaacca cgaaccgaac cctgcgcaac 1980

acctttgcgc ctagcgatca taaaatccgc cgcctccacc atttgtagaa aaatgtgacg 2040

aactcgtgag ctctgtacag tgaccggtga ctctttctgg catgcggaga gacggacgga 2100

cgcagagaga agggctgagt aataagccac tggccagaca gctctggcgg ctctgaggtg 2160

cagtggatga ttattaatcc gggaccggcc gcccctccgc cccgaagtgg aaaggctggt 2220

gtgcccctcg ttgaccaaga atctattgca tcatcggaga atatggagct tcatcgaatc 2280

accggcagta agcgaaggag aatgtgaagc caggggtgta tagccgtcgg cgaaatagca 2340

tgccattaac ctaggtacag aagtccaatt gcttccgatc tggtaaaaga ttcacgagat 2400

agtaccttct ccgaagtagg tagagcgagt acccggcgcg taagctccct aattggccca 2460

tccggcatct gtagggcgtc caaatatcgt gcctctcctg ctttgcccgg tgtatgaaac 2520

cggaaaggcc gctcaggagc tggccagcgg cgcagaccgg gaacacaagc tggcagtcga 2580

cccatccggt gctctgcact cgacctgctg aggtccctca gtccctggta ggcagctttg 2640

ccccgtctgt ccgcccggtg tgtcggcggg gttgacaagg tcgttgcgtc agtccaacat 2700

ttgttgccat attttcctgc tctccccacc agctgctctt ttcttttctc tttcttttcc 2760

catcttcagt atattcatct tcccatccaa gaacctttat ttcccctaag taagtacttt 2820

gctacatcca tactccatcc ttcccatccc ttattccttt gaacctttca gttcgagctt 2880

tcccacttca tcgcagcttg actaacagct accccgcttg agcagacatc accatgcctg 2940

aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc 3000

tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg 3060

gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc 3120

ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg 3180

agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg 3240

aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg 3300

ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca 3360

ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg 3420

tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg 3480

ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc 3540

tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt 3600

cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc 3660

agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt 3720

atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg 3780

atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg 3840

ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac 3900

tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa tagagtagat 3960

gccgaccgcg ggatccactt aacgttactg aaatcatcaa acagcttgac gaatctggat 4020

ataagatcgt tggtgtcgat gtcagctccg gagttgagac aaatggtgtt caggatctcg 4080

ataagatacg ttcatttgtc caagcagcaa agagtgcctt ctagtgattt aatagctcca 4140

tgtcaacaag aataaaacgc gttttcgggt ttacctcttc cagatacagc tcatctgcaa 4200

tgcattaatg cattgactgc aacctagtaa cgccttncag gctccggcga agagaagaat 4260

agcttagcag agctattttc attttcggga gacgagatca agcagatcaa cggtcgtcaa 4320

gagacctacg agactgagga atccgctctt ggctccacgc gactatatat ttgtctctaa 4380

ttgtactttg acatgctcct cttctttact ctgatagctt gactatgaaa attccgtcac 4440

cagcncctgg gttcgcaaag ataattgcat gtttcttcct tgaactctca agcctacagg 4500

acacacattc atcgtaggta taaacctcga aatcanttcc tactaagatg gtatacaata 4560

gtaaccatgc atggttgcct agtgaatgct ccgtaacacc caatacgccg gccgaaactt 4620

ttttacaact ctcctatgag tcgtttaccc agaatgcaca ggtacacttg tttagaggta 4680

atccttcttt ctagaagtcc tcgtgtactg tgtaagcgcc cactccacat ctccactcga 4740

gccctggagt ttttttttat cattttattt ctctatcatt gcccagcaaa tgttcgcgtg 4800

ggctgatgcc tggttactgt cttctttctt ggtgcttact ggcccttctg atggaggagc 4860

ctcgcgatat tgtttccttc atacccttct gtcattgatt tatacggcct tcaaaaatgt 4920

accataacat cttcaaggcg tgactcgttt ccaagggaag gttaattgtg gtcttgcaac 4980

gcgaaccgaa aatggactag ataggtcagg tgtcatctcg tacttcgctt cgggtgtcga 5040

ccgagtatcc gtagcactac attcctcatc tttaaagcca ttaggacaac gactagagtt 5100

aaccacaagc gctgctattg acatgacaca tcacagctca aaagcacaca tggacgtcgt 5160

tctaggtgca gcacctttcc cgcatgccgt ttcatacctc tcattgcctc atctactaag 5220

aacgaagcaa cagacttcgc agctgagcta accaatagtc tcgttatcga ccaaaagaag 5280

cctcatcttg gtctagatga taaaatcttg gctcaactgg atcggcaatt caagcacttt 5340

cctctgctgc cagcgagtcc ctcgtctccg actcgtcgcg ggtttgacca agaaggaaca 5400

aagctatgga atatttgcat gcaactcatg actgtctata gagacagaag tgaagacctt 5460

ctccttgcgt gcaaaggtac tactgtggga tcccaaggac acagactttc gctgagtaca 5520

gtatagtgaa ggcattcgct tacgcgatgt tggactatgc cgcaccatat caaggccagg 5580

gtaagtataa cacacttatc tattgtaagg attcgagaaa gctaatatta ccaggaagca 5640

acagggcctt agaggcagcc ttcagcatag caatgacgtg cataggtaag agcagatcct 5700

tgttgctttc tccaatttcc cttgcacgta gctctaacat cttgcatata gacaacgact 5760

gtctcagtct aagccagaaa atcatcgaag tcgcagctgt gcggttagat aaacttgaga 5820

gatatgaaag cgatgtcgaa aattctaagc ttcaacaata taccatcgag tactacatga 5880

ttcgtgccca cttggtatgg tctacccgtc tgtaataata cacatcagac tgacagctgg 5940

gacctaagta ggcctggctg cagggaagac tcgacatcgc agaacacttg ttctcgaaga 6000

taccagtatc tgataatggc aggggtcaag aacgtgtcat ggacatatgc tataagatcg 6060

gtaactgtgc gatttcacac aagcagtatg atgtctctat gaagtggcta gaaagagcgt 6120

tgagggcatg tgagtccatt aggcatatgc aacaggcatc gatactgtca aacaaggata 6180

aagagctctt gatacttcat gcttctggta gggtgctcct ggttgatgac aaagttaagt 6240

ttgttcacaa agaatagttc gtgccggcct tcatcttgat cctgaagagc acagtggctt 6300

cctttctgaa gctctggatg cattgaaatt cgtaagtctt gatatccagt tgttatatat 6360

tgctaatatt taacagcgtt acggtggcat gttccctgtc caggtcatcc aacttgagat 6420

gctggacaag gaaggagcag atgagattgt cttttcgcaa ggtgactgaa ttatagttcg 6480

tgattgtttt ccctcttcga ctgacataag agttaacagt gctacaaagt accattgaat 6540

cgcctgagct taaagactca cacctcgcaa tgtaatggcc aatatgaact cgtgtacgcc 6600

gtccggtact aacaaagtgg gtgtagcatc atgtattata tacagaagct gtttaataca 6660

tcgtgagcgc accggtcaca tctgtctgct catctgacaa gtaaccagtc ccgaggtctc 6720

catcagtatg ctcaagcaac 6740

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

aagaaaccca gacgatgttc 20

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cagcgcttgt ggttaactct 20

<210> 17

<211> 5388

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

taaaatccgc cgcctccacc atttgtagaa aaatgtgacg aactcgtgag ctctgtacag 60

tgaccggtga ctctttctgg catgcggaga gacggacgga cgcagagaga agggctgagt 120

aataagccac tggccagaca gctctggcgg ctctgaggtg cagtggatga ttattaatcc 180

gggaccggcc gcccctccgc cccgaagtgg aaaggctggt gtgcccctcg ttgaccaaga 240

atctattgca tcatcggaga atatggagct tcatcgaatc accggcagta agcgaaggag 300

aatgtgaagc caggggtgta tagccgtcgg cgaaatagca tgccattaac ctaggtacag 360

aagtccaatt gcttccgatc tggtaaaaga ttcacgagat agtaccttct ccgaagtagg 420

tagagcgagt acccggcgcg taagctccct aattggccca tccggcatct gtagggcgtc 480

caaatatcgt gcctctcctg ctttgcccgg tgtatgaaac cggaaaggcc gctcaggagc 540

tggccagcgg cgcagaccgg gaacacaagc tggcagtcga cccatccggt gctctgcact 600

cgacctgctg aggtccctca gtccctggta ggcagctttg ccccgtctgt ccgcccggtg 660

tgtcggcggg gttgacaagg tcgttgcgtc agtccaacat ttgttgccat attttcctgc 720

tctccccacc agctgctctt ttcttttctc tttcttttcc catcttcagt atattcatct 780

tcccatccaa gaacctttat ttcccctaag taagtacttt gctacatcca tactccatcc 840

ttcccatccc ttattccttt gaacctttca gttcgagctt tcccacttca tcgcagcttg 900

actaacagct accccgcttg agcagacatc accatgcctg aactcaccgc gacgtctgtc 960

gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc 1020

gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat 1080

agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg 1140

ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc 1200

tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt 1260

ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc 1320

gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata 1380

tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt 1440

gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc 1500

cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata 1560

acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac 1620

atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg 1680

aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt 1740

gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt 1800

cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc 1860

agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga 1920

cgccccagca ctcgtccgag ggcaaaggaa tagagtagat gccgaccgcg ggatccactt 1980

aacgttactg aaatcatcaa acagcttgac gaatctggat ataagatcgt tggtgtcgat 2040

gtcagctccg gagttgagac aaatggtgtt caggatctcg ataagatacg ttcatttgtc 2100

caagcagcaa agagtgcctt ctagtgattt aatagctcca tgtcaacaag aataaaacgc 2160

gttttcgggt ttacctcttc cagatacagc tcatctgcaa tgcattaatg cattgactgc 2220

aacctagtaa cgccttncag gctccggcga agagaagaat agcttagcag agctattttc 2280

attttcggga gacgagatca agcagatcaa cggtcgtcaa gagacctacg agactgagga 2340

atccgctctt ggctccacgc gactatatat ttgtctctaa ttgtactttg acatgctcct 2400

cttctttact ctgatagctt gactatgaaa attccgtcac cagcncctgg gttcgcaaag 2460

ataattgcat gtttcttcct tgaactctca agcctacagg acacacattc atcgtaggta 2520

taaacctcga aatcanttcc tactaagatg gtatacaata gtaaccatgc atggttgcct 2580

agtgaatgct ccgtaacacc caatacgccg gccgaaactt ttttacaact ctcctatgag 2640

tcgtttaccc agaatgcaca ggtacacttg tttagaggta atccttcttt ctagaagtcc 2700

tcgtgtactg tgtaagcgcc cactccacat ctccactcga cctgcaggca tgcaagcttg 2760

gcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat 2820

cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat 2880

cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc 2940

cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct 3000

gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg 3060

gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 3120

tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc 3180

ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt 3240

cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat 3300

ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg 3360

agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt 3420

tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga 3480

gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa 3540

gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt 3600

attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt 3660

gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc 3720

agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga 3780

ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat 3840

cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct 3900

gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc 3960

cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 4020

gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 4080

ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 4140

acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca 4200

ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta 4260

aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc 4320

aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa 4380

ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca 4440

ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta 4500

actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc 4560

caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca 4620

gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta 4680

ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag 4740

cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt 4800

cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc 4860

acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac 4920

ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac 4980

gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc 5040

tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat 5100

accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag 5160

cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac 5220

gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc 5280

actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt 5340

gtgagcggat aacaatttca cacaggaaac agctatgacc atgattac 5388

Claims (9)

1. The application of the recombinant Aspergillus Niger genetically engineered bacterium in the preparation of citric acid by fermentation is characterized in that the recombinant Aspergillus Niger genetically engineered bacterium is prepared by inactivating iron acquisition regulatory genes of Aspergillus Niger ATCC 12846; the nucleotide sequence of the inactivated iron acquisition regulatory gene is shown as SEQ ID NO. 2.

2. Use according to claim 1, characterized in that it comprises the following steps:

(1) Amplifying to obtain an upstream homology arm and a downstream homology arm of an iron acquisition regulatory gene by taking the genomic DNA of Aspergillus Niger ATCC12846 as a template;

(2) Amplifying to obtain a hygromycin resistance gene by taking the plasmid PAN7-1 as a template;

(3) Amplifying to obtain gene knockout fragments by taking the upstream homology arm and the downstream homology arm of the iron acquisition regulatory gene obtained in the step (1) and the hygromycin resistance gene obtained in the step (2) as templates;

(4) And (4) introducing the gene knockout fragment obtained in the step (3) into Aspergillus Niger ATCC12846 protoplasts for homologous recombination to obtain AcuM inactivated recombinant Aspergillus Niger genetically engineered bacteria.

3. Use according to claim 1, wherein the fermentation is an immobilized fermentation.

4. Use according to claim 3, wherein the immobilization medium of the immobilized fermentation is polyurethane.

5. The use according to claim 4, wherein the immobilization medium is present in an amount of 0.1-5 g/L fermentation medium.

6. Use according to claim 3, characterized in that the immobilization medium of the immobilized fermentation is pretreated sequentially with NaOH and HCl.

7. The use according to claim 1, wherein the fermentation medium is a fermentation medium obtained by enzymatic hydrolysis of corn meal with a liquefying enzyme, or prepared from bran extract.

8. The use according to claim 1, wherein the fermentation medium is a fermentation medium prepared from bran leachate, and the concentration of each component in the fermentation medium prepared from bran leachate is as follows: glucose 250 g/L, naNO ₃ 4 .2 g/L、KH ₂ PO ₄ 1.0g/L、MgSO ₄ ·7H ₂ O0.5 g/L, solvent is bran extract.

9. Use according to claim 1, wherein the temperature of the fermentation is 30-37 ℃.

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