CN109055419B - Construction method and application of recombinant microorganism with phosphorus-solubilizing activity - Google Patents

Abstract

The invention discloses a construction method and application of a recombinant microorganism with phosphorus-solubilizing activity. The method for constructing the recombinant microorganism with the phosphorus solubilizing activity comprises the step of introducing a coding gene of glucose dehydrogenase into a receptor microorganism to obtain the recombinant microorganism with the phosphorus solubilizing activity higher than that of the receptor microorganism. The invention constructs a recombinant microorganism with phosphorus-dissolving activity by endowing the activity of a receptor microorganism (bacillus megatherium WH320) glucose dehydrogenase CrGDH3A, wherein the phosphorus-dissolving capacity of the recombinant microorganism on tricalcium phosphate is 11.59 times that of the receptor microorganism, the phosphorus-dissolving capacity on aluminum phosphate is 16.23 times that of the corresponding receptor microorganism, and the phosphorus-dissolving capacity on ground phosphate rock is 14.00 times that of the corresponding receptor microorganism.

Description

Construction method and application of recombinant microorganism with phosphorus-solubilizing activity

The application is a divisional application with the name of 'engineering bacteria for expressing glucose dehydrogenase and construction method and application thereof' invented and created with the application number of 201611153688.2 and the application date of 2016, 12, and 14.

Technical Field

The invention relates to a construction method and application of a recombinant microorganism with phosphorus-solubilizing activity.

Background

In agricultural production, the increased application of phosphate fertilizer is a way of high input and low output. In China, approximately 2100 million to 2200 million tons of phosphate fertilizer are consumed each year, but the utilization rate of crops of the phosphate fertilizer in season is only 5 to 25 percent, and about 90 percent of the phosphate fertilizer is quickly chemically fixed after being applied to soil to form compounds such as calcium phosphate, iron, aluminum and the like with extremely low solubility. Phosphorus is a non-renewable resource, and with the continuous consumption of phosphorite reserves, China possibly faces the shortage of phosphorite and seriously restricts the grain production. Although the soil is not deficient in phosphorus, the soil is very low in effectiveness, and most of the soil is insoluble inorganic phosphorus, so that plants are difficult to absorb and utilize. Therefore, the activation of ineffective phosphorus in soil is one of the problems to be solved urgently in agricultural production.

Glucose Dehydrogenases (GDH), which belong to a family of short-chain alcohol dehydrogenases, catalyze the conversion of D-glucose to D-glucono-delta-lactone in the presence of a coenzyme, and the resulting D-glucono-delta-lactone is further spontaneously hydrolyzed to gluconic acid. The glucose dehydrogenase with high activity is researched and developed to further construct glucose dehydrogenase phosphorus-dissolving engineering bacteria, the capability of decomposing inorganic phosphorus in soil by phosphorus-dissolving bacteria can be obviously improved, and the method has great value in the aspects of activating ineffective phosphorus in soil, improving the utilization rate of phosphate fertilizer and reducing the investment of phosphate fertilizer.

Disclosure of Invention

The technical problem to be solved by the invention is how to construct the microorganism with the phosphorus dissolving activity.

In order to solve the above technical problems, the present invention provides a recombinant microorganism having a phosphate solubilizing activity.

The recombinant microorganism with the phosphorus-solubilizing activity is prepared by endowing a recipient microorganism with glucose dehydrogenase activity; the recombinant microorganism having a phosphorus solubilizing activity has a higher phosphorus solubilizing activity than the recipient microorganism.

In the above recombinant microorganism, the imparting of glucose dehydrogenase activity to the recipient microorganism is carried out by introducing a gene encoding glucose dehydrogenase into the recipient microorganism.

In order to solve the above technical problems, the present invention provides a method for constructing a recombinant microorganism having a phosphate solubilizing activity.

The method for constructing the recombinant microorganism with the phosphorus-solubilizing activity comprises the steps of introducing a coding gene of glucose dehydrogenase into a receptor microorganism to obtain the recombinant microorganism with the phosphorus-solubilizing activity, wherein the phosphorus-solubilizing activity of the recombinant microorganism is higher than that of the receptor microorganism; the glucose dehydrogenase is a protein of a) or b) or c) or d):

a) a protein consisting of an amino acid sequence shown in SEQ ID No. 2;

b) a protein consisting of an amino acid sequence shown as SEQ ID No. 6;

c) a fusion protein obtained by carboxyl terminal or/and amino terminal fusion protein label of the protein shown in a) or b);

d) the protein with the glucose dehydrogenase activity is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No.2 or SEQ ID No. 6.

In the present application, the phosphorus solubilizing activity refers to the ability to convert inorganic phosphorus into soluble phosphorus. Wherein, the soluble phosphorus refers to phosphorus which can be dissolved in water or dissolved in weak acid and then can be absorbed and utilized by plants. Soluble phosphorus includes water soluble phosphorus and/or exchangeable phosphorus. The inorganic phosphorus can be insoluble phosphate (such as tricalcium phosphate or aluminum phosphate) or ground phosphate rock.

In the above method, the name of the protein represented by a) is CrGDH 3A; SEQ ID No.2 consists of 796 amino acid residues.

In the above method, the protein shown in b) is named as CrGDH3A-His, the N end of CrGDH3A shown in SEQ ID No.2 is connected with MGSSHHHHHHSSGLVPRGSHM to obtain the fusion protein, and SEQ ID No.6 consists of 817 amino acid residues.

In the above method, the protein tag refers to a polypeptide or protein that is expressed by fusion with a target protein by using a DNA in vitro recombination technology, so as to facilitate expression, detection, tracing, and/or purification of the target protein.

The coding gene can be specifically the coding gene of glucose dehydrogenase shown in the following 1) or 2) or 3):

1) the coding sequence (CDS) is a DNA molecule shown in SEQ ID No.1 and is named as CrGDH3A gene;

2) the coding sequence is a DNA molecule shown in SEQ ID No.5 and is named as CrGDH3A-His gene;

3) a DNA molecule having 90% or more identity to the DNA molecule defined in 1) or 2) and encoding the glucose dehydrogenase.

In the context of the encoding gene, "identity" refers to sequence similarity to the native nucleic acid sequence. "identity" can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In the above recombinant microorganism or method, the recipient microorganism may be a prokaryotic microorganism.

In the above recombinant microorganism or method, the prokaryotic microorganism may be a gram-negative bacterium or a gram-positive bacterium.

In the above recombinant microorganism or method, the gram-negative bacterium may specifically be an Escherichia bacterium. The gram-positive bacterium may specifically be a bacterium of the genus bacillus.

In the above recombinant microorganism or method, the Escherichia bacterium may specifically be Escherichia coli. The bacillus bacteria may be bacillus megaterium.

In the recombinant microorganism or the method, the gene coding for glucose dehydrogenase may be introduced into the recipient microorganism via a recombinant expression vector pET-CrGDH 3A; the pET-CrGDH 3A is a recombinant expression vector obtained by replacing a fragment between NdeI and BamHI recognition sites of pET-28a (+) with a CrGDH3A gene shown in SEQ ID No. 1. pET-CrGDH 3A contains a CrGDH3A-His coding gene shown in SEQ ID No.5, and the amino acid sequence of protein CrGDH3A-His coded by the CrGDH3A-His coding gene is shown in SEQ ID No. 6. The CrGDH3A-His is a fusion protein obtained by connecting MGSSHHHHHHSSGLVPRGSHM to the N end of CrGDH3A shown in SEQ ID No. 2.

In the recombinant microorganism or the method, the gene encoding glucose dehydrogenase may be introduced into the recipient microorganism via a recombinant expression vector pWH-CrGDH 3A; the pWH-CrGDH3A is a recombinant expression vector obtained by forward replacing fragments among 2 BamHI recognition sites of pWH1520 by a CrGDH3A gene shown in SEQ ID No. 1. pWH-CrGDH3A contains CrGDH3A gene shown in SEQ ID No.1 in the sequence table, and pWH-CrGDH3A expresses protein CrGDH3A shown in SEQ ID No. 2.

The application of the recombinant microorganism or the method in dissolving inorganic phosphorus also belongs to the protection scope of the invention.

In the above application, the inorganic phosphorus may be insoluble phosphate (such as tricalcium phosphate or aluminum phosphate) or ground phosphate rock.

The biological material related to the glucose dehydrogenase is B1) or B2), B1) contains the expression cassette of the coding gene; B2) a recombinant vector containing the coding gene.

The recombinant vector in B2) may be, for example, pWH-CrGDH3A or pET-CrGDH 3A.

The application of the biological material in the preparation of glucose dehydrogenase also belongs to the protection scope of the invention.

The application of the biological material in preparing the recombinant microorganism with the phosphorus dissolving activity also belongs to the protection scope of the invention.

Experiments prove that in the prokaryotic expression of model bacteria (Escherichia coli is a receptor bacterium), glucose dehydrogenases CrGDH3A and CrGDH3A-His both have higher glucose dehydrogenase activity, and the enzyme activity of the glucose dehydrogenase CrGDH3A-His is 39.47 +/-1.03U/mg protein under the condition of 25 ℃ and pH7.8; in the phosphorus-solubilizing engineering bacteria (Bacillus megaterium WH320 is a receptor bacterium), the enzyme activity of glucose dehydrogenase CrGDH3A is 36.53 +/-1.16U/mg under the conditions of 40 ℃ and pH7.4. The invention constructs a recombinant microorganism with phosphorus-dissolving activity by endowing a receptor microorganism (bacillus megatherium WH320) with glucose dehydrogenase CrGDH3A activity, wherein the phosphorus-dissolving capacity of the recombinant microorganism on tricalcium phosphate is 11.59 times that of the receptor microorganism, the phosphorus-dissolving capacity on aluminum phosphate is 16.23 times that of the corresponding receptor microorganism, and the phosphorus-dissolving capacity on ground phosphate rock is 14.00 times that of the corresponding receptor microorganism. The invention provides important gene resources for cultivating new varieties of phosphorus-efficient crops by applying genetic engineering means and bioengineering bacteria for efficiently activating phosphorus nutrients in soil, and is beneficial to promoting the phosphorus-dissolving engineering bacteria to move from a laboratory research and development stage to a field application stage.

Drawings

FIG. 1 is a physical map of pET-CrGDH 3A and pET-CrGDH 3B. Wherein gdh3 is CrGDH3A gene or CrGDH3B gene.

FIG. 2 is an SDS-PAGE pattern of the induction expression of glucose dehydrogenase in E.coli. Wherein, 1: a protein Marker; 2: escherichia coli BL21(DE 3); 3: pET-CrGDH 3A/BL 21; 4: pET-30a (+)/BL 21; 5: pET-CrGDH3B/BL 21. The arrows indicate the destination strips.

FIG. 3 is a physical map of pWH-CrGDH 3A. Wherein gdh3gene is CrGDH3A gene.

FIG. 4 is an SDS-PAGE pattern of glucose dehydrogenase expressed in the glucose dehydrogenase-engineering bacteria. Wherein, M: a protein Marker; 1: intracellular precipitation; 2: intracellular supernatant; 3: extracellular supernatant.

FIG. 5 is a graph showing the effect of pH on the enzymatic activity of glucose dehydrogenase expressed in a glucose dehydrogenase-engineering bacterium.

FIG. 6 is a graph showing the effect of temperature on the enzymatic activity of glucose dehydrogenase expressed in a glucose dehydrogenase-engineering bacterium.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation and functional verification of glucose dehydrogenase CrGDH3

Construction of recombinant expression vector

In order to improve the activity of glucose dehydrogenase, glucose dehydrogenase CrGDH3A (abbreviated as 3A) is obtained by substituting an amino acid residue for a Citrobacter rodentium-derived glucose dehydrogenase (hereinafter referred to as CrGDH3B, abbreviated as 3B) shown in Genbank Access Number WP-012904518. The amino acid sequence of CrGDH3A is SEQ ID No.2, the amino acid sequence of CrGDH3B is SEQ ID No.4, and the different amino acid residues of CrGDH3A and CrGDH3B are shown in Table 1 and Table 2.

TABLE 1, CrGDH3A and CrGDH3B differential amino acid residues

TABLE 2 CrGDH3A and CrGDH3B differential amino acid residues

The CrGDH3A gene shown in SEQ ID No.1 and the CrGDH3B gene shown in SEQ ID No.3 were prepared separately.

A recombinant expression vector was obtained by replacing the NdeI and BamHI recognition sites of pET-28a (+) (EMD Biosciences, available from New York, Beijing, and having a size of 5369bp) with CrGDH3A gene shown in SEQ ID No.1, while keeping the other sequence of pET-28a (+) unchanged, and was designated as pET-CrGDH 3A (FIG. 1). pET-CrGDH 3A contains His label fusion protein CrGDH3A-His coding gene shown in SEQ ID No.5, and the amino acid sequence of protein CrGDH3A-His coded by CrGDH3A-His coding gene is shown in SEQ ID No. 6. The CrGDH3A-His is a fusion protein obtained by connecting MGSSHHHHHHSSGLVPRGSHM to the N end of CrGDH3A shown in SEQ ID No. 2.

A recombinant expression vector was obtained by replacing the NdeI and BamHI recognition sites of pET-28a (+) (EMD Biosciences, available from New York, Beijing, and having a size of 5369bp) with CrGDH3B gene shown in SEQ ID No.3, while keeping the other sequence of pET-28a (+) unchanged, and was designated as pET-CrGDH3B (FIG. 1). pET-CrGDH3B contains His label fusion protein CrGDH3B-His coding gene shown in SEQ ID No.7, and the amino acid sequence of protein CrGDH3B-His coded by CrGDH3B-His coding gene is shown in SEQ ID No. 8. CrGDH3B-His is a fusion protein obtained by connecting MGSSHHHHHHSSGLVPRGSHM to the N end of CrGDH3B shown in SEQ ID No. 4.

Preparation of recombinant Escherichia coli for expressing glucose dehydrogenase

1. Expression of CrGDH3A-His

Transforming Escherichia coli BL21(DE3) (Tiangen company) with the calcium chloride method for pET-CrGDH 3A in the first step, screening and culturing positive clones by kanamycin resistance screening, picking up single clones, carrying out PCR identification by taking P1 (5'-ATGGCTATTAACAATACAGGCTC-3') and P2 (5'-TTATTTCACATCATCCGGCAGCG-3') as primers, and taking the positive clones which are subjected to PCR identification to obtain 2391bp PCR products as genetically engineered bacteria, wherein the positive clones are named as pET-CrGDH 3A/BL 21. pET-CrGDH 3A/BL21 strain was picked up, inoculated into LB medium containing 100ug/ml kanamycin (a medium obtained by adding kanamycin to LB medium to 100. mu.g/ml kanamycin concentration), and cultured at 37 ℃ to 0D₆₀₀When the value (LB medium containing 100. mu.g/ml kanamycin as blank control) reached 0.6, IPTG was added to the final concentration of l mM, induction was carried out at 28 ℃ for 6 hours at a rotation speed of 150r/min, the culture was collected, centrifuged at 4000r/min for 20 minutes, and then the cells were resuspended with 50mM Tris-HCl (pH7.1) to obtain a cell content of 10⁸cfu/ml of a cell suspension, ultrasonication of the cell suspension for 30min (50% power, 10s of work, 20s of pause), addition of Triton-X100 to the disrupted cell suspension to a final concentration of 1%, leaching overnight at 4 ℃, centrifugation at 12000r/min for 10min, collection of the supernatant (containing the total cell proteins), and designation of the supernatant as a crude enzyme solution of CrGDH 3A-His.

2. Expression of CrGDH3B-His

Transforming the pET-CrGDH3B of the second step into Escherichia coli BL21(DE3) by the calcium chloride method (Tiangen Co.), screening and culturing positive clones by kanamycin resistance screening, picking out single clones, and using P3 (5'-ATGGCTGAAAACAATGCACG-3') and P4 (5)'-TTACTTCTCGTCGTCCGGCA-3') as a primer, performing PCR identification, and taking a positive clone of the PCR product of 2391bp obtained by the PCR identification as a genetic engineering bacterium, which is named as pET-CrGDH3B/BL 21. The pET-CrGDH3B/BL21 strain was picked up, inoculated into LB medium containing 100. mu.g/ml kanamycin (a medium obtained by adding kanamycin to LB medium to 100. mu.g/ml kanamycin concentration), and cultured at 37 ℃ to 0D₆₀₀When the value (LB medium containing 100. mu.g/ml kanamycin as blank control) reached 0.6, IPTG was added to the final concentration of l mM, induction was carried out at 28 ℃ for 6 hours at a rotation speed of 150r/min, the culture was collected, centrifuged at 4000r/min for 20 minutes, and then the cells were resuspended with 50mM Tris-HCl (pH7.1) to obtain a cell content of 10⁸cfu/ml of a cell suspension, ultrasonication of the cell suspension for 30min (50% power, 10s of work, 20s of pause), addition of Triton-X100 to the disrupted cell suspension to a final concentration of 1%, leaching overnight at 4 ℃, centrifugation at 12000r/min for 10min, collection of the supernatant (containing the total cell proteins), and designation of the supernatant as a crude enzyme solution of CrGDH 3B-His.

3. Empty vector control bacterium

pET-28a (+) was transformed into E.coli BL21(DE3) in the same manner as in step 1, and the resulting recombinant E.coli was named pET-28a (+)/BL 21. Using pET-28a (+)/BL21 as an empty vector control, total cell protein was prepared by induction expression according to the method described in step 1 above. The pET-28a (+)/BL21 strain was selected, inoculated in LB medium containing 100. mu.g/ml kanamycin (a medium obtained by adding kanamycin to LB medium to 100. mu.g/ml kanamycin concentration), and cultured at 37 ℃ to 0D₆₀₀When the value (LB medium containing 100. mu.g/ml kanamycin as blank control) reached 0.6, IPTG was added to the final concentration of l mM, induction was carried out at 28 ℃ for 6 hours at a rotation speed of 150r/min, the culture was collected, centrifuged at 4000r/min for 20 minutes, and then the cells were resuspended with 50mM Tris-HCl (pH7.1) to obtain a cell content of 10⁸cfu/ml thallus suspension, ultrasonic crushing the thallus suspension for 30min (50% power, 10s of work and 20s of pause), adding Triton-X100 into the crushed cell suspension to the final concentration of 1%, leaching at 4 ℃ overnight, centrifuging at 12000r/min for 10min, collecting supernatant (containing total thallus protein), and naming the supernatant as the empty vector control bacteria crude enzyme solution.

4. Blank control bacterium Escherichia coli BL21(DE3)

Coli BL21(DE3) was used as a blank control, and total cell protein was prepared by induction expression according to the method described in step 1. Escherichia coli BL21(DE3) strain was selected, inoculated into LB medium, and cultured at 37 ℃ to 0D₆₀₀When the value (LB medium as blank control) reaches 0.6, adding IPTG to final concentration l mM, inducing at 28 deg.C for 6h at 150r/min, collecting culture solution, centrifuging at 4000r/min for 20min, and re-suspending the thallus with 50mM Tris-HCl (pH7.1) to obtain thallus content of 10⁸cfu/ml of thallus suspension, ultrasonically crushing the thallus suspension for 30min (50% power, 10s of work and 20s of pause), adding Triton-X100 into the crushed cell suspension to the final concentration of 1%, leaching at 4 ℃ overnight, centrifuging at 12000r/min for 10min, collecting supernatant (containing total thallus protein), and naming the supernatant as blank control crude enzyme solution.

30. mu.L of CrGDH3A-His crude enzyme solution (10-derived from)⁸cfu/ml pET-CrGDH 3A/BL21), 30. mu.L CrGDH3B-His crude enzyme solution (from 10⁸cfu/ml pET-CrGDH3B/BL 21), 30. mu.L of crude enzyme solution of empty vector control bacteria (from 10⁸cfu/ml pET-28a (+)/BL21) and 30. mu.L of the placebo crude enzyme solution (from 10⁸cfu/ml E.coli BL21(DE3)) was analyzed by SDS-PAGE on the same gel (gel concentration of 12%) with a uniform pore volume and shape and a pore volume of 80. mu.L.

The SDS-PAGE results are shown in FIG. 2, which shows that although the crude enzyme solution of CrGDH3A-His, the crude enzyme solution of CrGDH3B-His, the crude enzyme solution of empty vector control bacteria and the crude enzyme solution of blank control bacteria all have 87kD bands, the content of the 87kD polypeptide in the crude enzyme solution of CrGDH3A-His and the crude enzyme solution of CrGDH3B-His is obviously higher than that of the 87kD polypeptide in the crude enzyme solution of empty vector control bacteria and the crude enzyme solution of blank control bacteria, and the content of the 87kD polypeptide in the crude enzyme solution of CrGDH3A-His is higher than that of the 87kD polypeptide in the crude enzyme solution of CrGDH 3B-His. The results show that both CrGDH3A-His and CrGDH3B-His are expressed in Escherichia coli BL21(DE3), and the expression level of CrGDH3A-His in Escherichia coli BL21(DE3) is obviously higher than that of CrGDH3B-His in Escherichia coli BL21(DE 3).

Third, the catalytic activity of the glucose dehydrogenase of CrGDH3A-His and CrGDH3B-His was measured

Respectively purifying the crude enzyme solution of the CrGDH3A-His, the crude enzyme solution of the CrGDH3B-His, the crude enzyme solution of the empty vector control bacteria and the crude enzyme solution of the blank control bacteria by using a nickel column (a high-affinity Ni-NTA Rasin product from American general company), pretreating the nickel column, adding the crude enzyme solution, and then adding an imidazole-containing eluent (50mM NaH)₂PO₄300mM NaCl, 250mM imidazole, pH8.0) at 4 ℃ for 10min, centrifuging at 3000rpm for 1min, collecting the eluate, repeating the elution once, collecting the eluate, and taking 1ml of the eluate for SDS-PAGE analysis. The sequence determination result of CrGDH3A-His shows that the 15 amino acids at the N terminal are the 1 st to 15 th amino acids of the sequence 2 in the sequence table, and the sequence determination result of CrGDH3B-His shows that the 15 amino acids at the N terminal are the 1 st to 15 th amino acids of the sequence 4 in the sequence table.

Dialyzing the collected eluent by using double distilled water, and desalting and ionizing to respectively obtain pure CrGDH3A-His enzyme liquid, pure CrGDH3B-His enzyme liquid, pure empty vector control bacterium enzyme liquid and pure blank control bacterium enzyme liquid as enzyme liquids to be detected. And (4) quantitatively determining the protein content of the enzyme solution to be detected by using a BCA protein quantitative kit.

The activity of glucose dehydrogenase is measured by taking glucose as a substrate and carrying out colorimetric quantitative analysis on the activity of the glucose dehydrogenase according to the generation of red. To 50. mu.L of the enzyme solution to be tested (pure CrGDH3A-His enzyme solution, pure CrGDH3B-His enzyme solution, pure empty vector control bacteria enzyme solution or pure blank control bacteria enzyme solution) was added 50. mu.L of a buffer solution (PQQ (pyrroloquinoline quinone) and CaCl were added to 100mmol/L of MOPS buffer solution) of pH7.8₂The PQQ content was adjusted to 10. mu. mol/L and CaCl₂Liquid obtained with the content of 2 mol/L), and is pretreated for 1 hour at 37 ℃ to stabilize the structure of the enzyme, so as to obtain pretreated enzyme liquid. Then, 1mL of 50mmol/L Tris-HCl buffer solution with pH7.8 is added into the cuvette, then 100 μ L of 20mmol/L phenazine methyl sulfate solution, 6.7 mmol/L2, 6-dichloroindophenol sodium (DCIP) solution and 1mol/L glucose solution are respectively added, after uniform mixing, 50 μ L of pretreatment enzyme solution is added, finally the volume is fixed to 3mL, the reaction temperature is 25 ℃, and the change of the absorbance value under 600nm per minute is measured. The enzyme activity unit (U) is defined as:an amount of an enzyme capable of oxidizing 1. mu. mol of glucose (or reducing 1. mu. mol of DCIP) within 1min at 25 ℃ and pH 7.8. The specific activity of glucose dehydrogenase is calculated as the activity of the enzyme per unit total protein in U/mg.

The experiment was performed in triplicate. The results show that the pure empty vector control bacterial enzyme solution and the pure blank control bacterial enzyme solution have no glucose dehydrogenase activity, the enzyme activity of the glucose dehydrogenase of CrGDH3A-His expressed by pET-CrGDH 3A/BL21 is 39.47 +/-1.03U/mg protein, and the enzyme activity of the glucose dehydrogenase of CrGDH3B-His expressed by pET-CrGDH3B/BL21 is 7.39 +/-0.26U/mg protein. The enzyme activity of the CrGDH3A-His glucose dehydrogenase is 5.34 times of that of the CrGDH3B-His glucose dehydrogenase. The yield of glucose dehydrogenase of pET-CrGDH 3A/BL21 is 25.65/10⁸The yield of the glucose dehydrogenase of cfu pET-CrGDH 3A/BL21 and pET-CrGDH3B/BL21 is 5.09U/10⁸cfu pET-CrGDH3B/BL 21. The yield of the glucose dehydrogenase of pET-CrGDH 3A/BL21 is 5 times that of pET-CrGDH3B/BL 21.

Example 2 cultivation of recombinant microorganism having phosphorus-solubilizing Activity-glucose dehydrogenase phosphorus-solubilizing engineering bacterium and functional identification thereof

1. Construction of shuttle expression vector of glucose dehydrogenase CrGDH3A gene

In order to obtain a phosphate solubilizing engineering bacterium for efficiently expressing a glucose dehydrogenase CrGDH3A gene, a shuttle expression vector capable of cross-host expression needs to be constructed first. The pWH1520 expression vector (7929bp) is a bacillus megaterium efficient shuttle expression vector (product of MoBiTec company, Germany, purchased from Beijing Byleldi Biotechnology company), carries a BamHI enzyme cutting site at the downstream of the xylA promoter, has ampicillin and tetracycline resistance genes, and can stably express target protein.

The sequence of the CrGDH3A gene was analyzed by using DNAMAN software to find that no BamHI cleavage site was present, and primers were designed based on the complete coding region sequence of the CrGDH3A gene, and Bam HI cleavage sites (GGATCC) were added to both the upstream and downstream primers. The upstream and downstream primers are respectively: p5: 5' -ATGGATCCATGGCTATTAACAATACAGGCTC-3' and P6: 5' -GCGGATCCTTATTTCACATCATCCGGCAGCG-3'). Using pET-CrGDH 3A of step one as a template, using P5 and P6 as primers, and usingIntroducing BamHI enzyme recognition sites into 5 'end and 3' end of complete coding region of CrGDH3A gene respectively to obtain PCR product of CrGDH3A gene with enzyme recognition sites; the PCR product of the shuttle expression vector pWH1520 and CrGDH3A gene with an enzyme recognition site was digested with BamHI, and the recovered digested product was digested with T₄And (3) ligase ligation, screening positive clones after transformation of ligation products, and sequencing. Because the gene is connected to the expression vector after single enzyme digestion, a PCR (polymerase chain reaction) and sequencing comparison method are combined to screen positive bacterial plaques of the CrGDH3A gene which is positively inserted into the shuttle expression vector pWH1520, and recombinant plasmids are extracted to finally obtain the shuttle expression vector of the CrGDH3A gene. The recombinant expression vector, which was obtained by forward substitution of the fragment between 2 BamHI recognition sites of pWH1520 with CrGDH3A gene shown in SEQ ID No.1 as a result of sequencing, was designated as pWH-CrGDH3A (FIG. 3). pWH-CrGDH3A contains CrGDH3A gene shown in SEQ ID No.1 in the sequence table, and pWH-CrGDH3A expresses protein CrGDH3A shown in SEQ ID No. 2.

2. Obtaining of glucose dehydrogenase engineering bacteria and enzymological characteristics of CrGDH3A expressed by the same

The recombinant vector pWH-CrGDH3A is transferred into Bacillus megaterium (WH320, purchased from Shanghai Beino Biotech Co.) by protoplast transformation method to obtain the glucose dehydrogenase engineering bacteria. The glucose dehydrogenase engineering bacteria were inoculated into LB medium containing tetracycline (medium obtained by adding tetracycline to LB medium to a tetracycline concentration of 100. mu.g/ml), and cultured overnight. Transferring 2% inoculum size to the LB culture medium containing tetracycline, continuously culturing to logarithmic phase, adding xylose to final concentration of 0.5%, inducing and culturing for 6h, centrifuging at 4000r/min at room temperature for 15min, and collecting supernatant as extracellular supernatant; collecting the precipitate, adding 2 times volume of phosphate buffer (pH6.0), and disrupting the cells to obtain a disrupted cell suspension. Adding Triton-X100 to the crushed cell suspension to a final concentration of 1%, leaching at 4 ℃ overnight, and centrifuging at 12000r/min for 10min to obtain the supernatant as intracellular supernatant and precipitate as intracellular precipitate. And performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis and enzyme activity determination on the intracellular supernatant, the intracellular sediment and the extracellular supernatant respectively. And (4) quantitatively determining the protein content of the enzyme solution to be detected by using a BCA protein quantitative kit.

The activity of glucose dehydrogenase CrGDH3A was measured according to the method of example 1, and the experiment was repeated three times. SDS-PAGE (FIG. 4) shows that the gene CrGDH3A can be normally expressed in the glucose dehydrogenase engineering bacteria, the expression product is intracellular protein (intracellular supernatant lane), and the molecular weight of the expression product is about 87 kD. The glucose dehydrogenase activity of CrGDH3A expressed by the glucose dehydrogenase engineering bacteria is 33.85 +/-1.53U/mg.

3. Effect of pH on the catalytic Activity of glucose dehydrogenase engineering bacteria

The glucose dehydrogenase engineering bacteria of step 2 were inoculated into LB medium containing ampicillin and tetracycline (medium obtained by adding ampicillin and tetracycline to LB medium until the concentration of ampicillin and tetracycline were both 100. mu.g/ml) and cultured overnight. Transferring the strain with the inoculum size of 2% into the LB culture medium containing tetracycline to continue culturing to logarithmic phase, adding xylose to the final concentration of 0.5%, performing induced culture for 6h, centrifuging at the room temperature at the speed of 4000r/min for 15min, collecting precipitate, adding 2 times volume of phosphate buffer (pH7.0), and crushing cells to obtain a crushed cell suspension. Adding Triton-X100 into the cell disruption suspension to a final concentration of 1%, leaching at 4 ℃ overnight, and centrifuging at 12000r/min for 10min to obtain supernatant as crude glucose dehydrogenase enzyme solution as enzyme solution to be detected.

10 reaction systems with different pH values are adopted: a citric acid-phosphoric acid buffer solution system (a pH5.4 reaction system, a pH5.8 reaction system, a pH6.2 reaction system, a pH6.6 reaction system, and a pH7.0 reaction system); the Tris buffer solution system (pH7.4 reaction system, pH7.8 reaction system, pH8.2 reaction system, pH8.6 reaction system and pH9.0 reaction system) is used for determining the capability of the glucose dehydrogenase engineering bacteria in catalyzing the dehydrogenation of glucose.

The 10 reaction systems with different pH values consist of enzyme solution to be detected, phenazine methyl sulfate, 2, 6-dichloroindophenol sodium (DCIP), glucose and corresponding buffer solutions.

50 μ L of buffer solution with corresponding pH value is added into 50 μ L of enzyme solution to be detected (PQQ (pyrroloquinoline quinone) and CaCl are added into 100mmol/L MOPS buffer solution₂The PQQ content was adjusted to 10. mu. mol/L and CaCl₂Obtained in an amount of 2mol/LLiquid), and pretreating at 37 ℃ for 1 hour to stabilize the structure of the enzyme to obtain pretreated enzyme liquid.

Respectively adding 1mL of buffer solutions with different pH values and 50mmol/L into a cuvette, then respectively adding 100 mu L of each of phenazine methyl sulfate 20mmol/L, 2, 6-dichloroindophenol sodium (DCIP) 6.7mmol/L and glucose 1mol/L, uniformly mixing, adding 50 mu L of pretreatment enzyme solution, finally fixing the volume to 3mL, measuring the change of the absorbance value under 600nm per minute at the reaction temperature of 25 ℃. The enzyme activity unit (U) is defined as: an amount of enzyme capable of oxidizing 1. mu. mol of glucose (or reducing 1. mu. mol of DCIP) within 1min at 25 ℃. The specific activity of glucose dehydrogenase is calculated as the activity of the enzyme per unit total protein, the unit is U/mg, the highest enzyme activity is taken as 100%, and the relative activity is converted. The experiment was repeated three times.

The optimum pH of the catalytic activity of the glucose dehydrogenase-engineered bacterium was 7.4, the enzyme activity at pH 6.6-7.8 was 80% of the optimum pH, the enzyme activity was about 20% of the optimum pH at pH5.4 and pH5.8, and the enzyme activity was less than 15% of the optimum pH at pH8.6 and pH9 (FIG. 5).

4. Effect of temperature on the catalytic Activity of glucose dehydrogenase engineering bacteria

And (3) inoculating the glucose dehydrogenase engineering bacteria in the step (2) into the LB culture medium containing ampicillin and tetracycline, and culturing overnight. Transferring the strain with the inoculum size of 2% into the LB culture medium containing tetracycline to continue culturing to logarithmic phase, adding xylose to the final concentration of 0.5%, performing induced culture for 6h, centrifuging at the room temperature at the speed of 4000r/min for 15min, collecting precipitate, adding 2 times volume of phosphate buffer (pH7.0), and crushing cells to obtain a crushed cell suspension. Adding Triton-X100 into the cell disruption suspension to a final concentration of 1%, leaching at 4 ℃ overnight, and centrifuging at 12000r/min for 10min to obtain supernatant as crude glucose dehydrogenase enzyme solution as enzyme solution to be detected. To 50. mu.L of the enzyme solution to be assayed, 50. mu.L of a buffer solution having pH7.4 (PQQ (pyrroloquinoline quinone) and CaCl were added to 100mmol/L of MOPS buffer solution)₂The PQQ content was adjusted to 10. mu. mol/L and CaCl₂Liquid obtained with the content of 2 mol/L), and is pretreated for 1 hour at 37 ℃ to stabilize the structure of the enzyme, so as to obtain pretreated enzyme liquid. In a Tris buffer system (pH7.4) inThe ability of the glucose dehydrogenase engineering bacteria to catalyze glucose is measured within the temperature range of 20-70 ℃. The reaction system consists of enzyme solution to be detected, phenazine methyl sulfate, 2, 6-dichloroindophenol sodium (DCIP), glucose and Tris buffer solution (pH7.4) liquid system. Adding 1mL of 50mmol/L Tris buffer solution (pH7.4) into a cuvette respectively, then adding 100 mu L each of 20mmol/L phenazine methyl sulfate, 6.7 mmol/L2, 6-dichloroindophenol sodium (DCIP) and 1mol/L glucose respectively, mixing uniformly, adding 50 mu L of pretreatment enzyme solution, finally fixing the volume to 3mL, and measuring the change of absorbance value under 600nm per minute. The enzyme activity unit (U) is defined as: an amount of enzyme capable of oxidizing 1. mu. mol of glucose (or reducing 1. mu. mol of DCIP) within 1min at a corresponding temperature of pH 7.4. The specific activity of glucose dehydrogenase is calculated as the activity of the enzyme per unit total protein in U/mg.

The optimum reaction temperature of the glucose dehydrogenase CrGDH3A induced and expressed by the glucose dehydrogenase engineering bacteria in step 2 is 40 ℃, the activity of the enzyme reaches 36.53 +/-1.16U/mg, the activity of the enzyme is maintained at a high level in the range of 30-45 ℃, the activity of the enzyme shows a trend of rapidly decreasing after 50 ℃, and the activity of the enzyme is difficult to detect when the temperature exceeds 70 ℃ (FIG. 6).

5. Phosphorus dissolving effect of glucose dehydrogenase engineering bacteria

Respectively inoculating the glucose dehydrogenase engineering bacteria and the bacillus megaterium WH320 (receptor bacteria) in the step 2 into a phosphate rock powder liquid culture medium, a tricalcium phosphate liquid culture medium and an aluminum phosphate liquid culture medium, so that the contents of the glucose dehydrogenase engineering bacteria and the bacillus megaterium WH320 are 10⁸cfu/mL, at 37 degrees C culture to logarithmic growth phase, adding xylose to the final concentration of 0.5%, 37 degrees C160 r/min shaking culture, inoculating tricalcium phosphate culture medium and aluminum phosphate culture medium in the 7 th day culture liquid, and inoculating phosphate rock powder culture medium in the 14 th day culture liquid. Centrifuging at 10000r/min at 4 deg.C for 10min, collecting supernatant, directly determining water soluble phosphorus (also called available phosphorus or available phosphorus) content in culture solution of inoculated glucose dehydrogenase engineering bacteria and Bacillus megaterium WH320 (acceptor bacteria) at wavelength of 700nm by using molybdenum-antimony colorimetric method and type 722 spectrophotometer, setting corresponding Control (CK) of non-inoculated bacteria, and providing effective Control (CK) belowPhosphorus content is the value of the corresponding Control (CK) minus no inoculation, and the test is repeated 3 times.

Wherein, the pH value of the ground phosphate rock liquid culture medium is 7.0, and the preparation method comprises the following steps: sterilizing the culture solution with water as solvent and the following solutes at 115 deg.C for 30 min: glucose 5g/L, xylose 5g/L, NaCl 0.2g/L, MgSO₄·7H₂O 0.1g/L，KCl 0.2g/L，(NH₄)₂SO₄0.5g/L, yeast extract 0.5g/L, 5g phosphate rock powder (30% P from Yunnan Chengjiang Dongtai phosphate fertilizer Co., Ltd.)₂O₅Content, 13% phosphorus content), distilled water was added to 1000 ml.

The pH value of the tricalcium phosphate liquid culture medium is 7.0, and the preparation method comprises the following steps: sterilizing the culture solution with water as solvent and the following solutes at 115 deg.C for 30 min: glucose 5g/L, xylose 5g/L, NaCl 0.2g/L, MgSO₄·7H₂O 0.1g/L，KCl 0.2g/L，(NH₄)₂SO₄0.5g/L, yeast extract 0.5g/L, tricalcium phosphate 5.0g/L, and distilled water to 1000 ml.

The pH value of the aluminum phosphate liquid culture medium is 7.0, and the preparation method comprises the following steps: sterilizing the culture solution with water as solvent and the following solutes at 115 deg.C for 30 min: glucose 5g/L, xylose 5g/L, NaCl 0.2g/L, MgSO₄·7H₂O 0.1g/L，KCl 0.2g/L，(NH₄)₂SO₄0.5g/L, yeast extract 0.5g/L, aluminum phosphate 5.0g/L, and distilled water to 1000 ml.

The results show that the content of available phosphorus of the culture solution of the glucose dehydrogenase engineering bacteria of the step 2 cultured in a tricalcium phosphate liquid culture medium for 7 days is 143.15 +/-7.16 mu mol/L, the content of available phosphorus of the culture solution cultured in an aluminum phosphate liquid culture medium for 7 days is 78.90 +/-3.95 mu mol/L, and the content of available phosphorus of the culture solution cultured in a phosphorite powder liquid culture medium for 14 days is 34.57 +/-2.07 mu mol/L; the content of available phosphorus in a culture solution of the bacillus megaterium WH320 serving as a recipient bacterium cultured in a tricalcium phosphate liquid culture medium for 7 days is 12.35 +/-0.62 mu mol/L, the content of available phosphorus in a culture solution cultured in an aluminum phosphate liquid culture medium for 7 days is 4.86 +/-0.27 mu mol/L, and the content of available phosphorus in a culture solution cultured in a phosphorite powder liquid culture medium for 14 days is 2.47 +/-0.12 mu mol/L. As can be seen, the phosphorus-dissolving capacity of the glucose dehydrogenase engineering bacteria in the step 2 to tricalcium phosphate is 11.59 times that of bacillus megaterium WH320 serving as a receptor bacteria, the phosphorus-dissolving capacity to aluminum phosphate is 16.23 times that of bacillus megaterium WH320 serving as the receptor bacteria, and the phosphorus-dissolving capacity to ground phosphate rock is 14.00 times that of bacillus megaterium WH320 serving as the receptor bacteria.

<110> institute of agricultural resources and agricultural regionalism of Chinese academy of agricultural sciences

<120> construction method and application of recombinant microorganism with phosphorus-solubilizing activity

<130> GNCFH181846

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 2391

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggctatta acaatacagg ctcgcgacga ctcctcgtgg tgttaacggc cctctttgca 60

gctctttgcg ggctgtatct tctcattggc ggaggctggt tggtcgccat tggcggctcc 120

tggtactacc cgatcgccgg tctggcgatg ctgggcgtcg cctggctgct gtggcgcagc 180

aaacgttccg cactctggct gtacgccgcc ctgctcctcg ccaccctgat ttggggcgtg 240

tgggaagttg gtttcgactt ctgggcgctg actccgcgca gcgacattct ggtcttcttc 300

ggcatctggc tgatcctgcc gtttgtctgg cgtcgcctgg tcattcctgc cagcggcgca 360

gttgccgcac tggtggtcgc gctgttgatt agcggtggta tcctcacctg ggcgggcttc 420

aacgacccgc aggagatcga cggcgcgctc agcgcggagt cgacgcctgc acaggccatc 480

tcaccagtgg ctgacggcga ctggccggcg tatggccgca atcaggaagg tcaacgcttt 540

tcaccactga agcaaattca cgccgataac gtccacaagc tgaaagaagc ctgggtgttc 600

cgtactggcg atgtgaagca gccgaacgat ccgggtgaaa tcaccaatga agtgacgcca 660

attaaagtgg gcgacacgct gtatctgtgc accgctcacc agcgtctgtt cgcgctggag 720

gcggcgacgg gtaaagaaaa atggcattac gatcctgagc tgaaaaccaa cgagtctttc 780

cagcatgtaa cctgccgtgg tgtctcttat catgaagcca aagcagaaac tgcttcgccg 840

gaagtgatgg cggattgccc gcgtcgtatc attctcccgg tcaatgatgg ccgcctgatt 900

gcgattaacg ctgaaaacgg caagctgtgc gaaaccttcg ctaataaagg cgtgctcaat 960

ctgcaaagca atatgccaga caccaaaccg ggtctgtatg agccgacttc gccgccgatt 1020

atcaccgata aaacgattgt gattgctggt tcagtaacgg ataacttctc cacccgcgaa 1080

acctcgggcg tgatccgtgg ttttgacgtc aataacggta aactgctgtg ggcgttcgat 1140

ccgggtgcga aagacccgaa tgcaatccct tccgatgagc actcttttac ctttaactcg 1200

ccgaactcct gggcgccagc ggcctatgac gcgaagctgg acctcgttta cctgccgatg 1260

ggggtctcga cgccggatat ctggggcgga caccggacgc cggagcagga gcgctacgcc 1320

agttccattc tggcgctgaa cgcgaccacc ggtaaactcg cctggagcta tcagacggtt 1380

caccacgatc tgtgggatat ggacatgccg tcccagccga cgctggcgga tattaccgtc 1440

aacggtgaga aagtcccggt tatctacgcg ccagcgaaaa ccggtaacat ctttgtcctc 1500

gaccgccgta acggcgagct ggtcgttcct gcaccggaaa aaccggttcc gcagggggcc 1560

gcgaaaggcg attacgttac ccctactcaa ccgttctctg agctgagctt ccgtccgaca 1620

aaagatctaa gcggtgcgga tatgtggggt gccaccatgt ttgaccaact ggtgtgccgc 1680

gtgatgttcc accagatgcg ctatgaaggc attttcaccc caccatctga acagggtacg 1740

ctggtcttcc cgggtaacct ggggatgttc gaatggggcg gtatttcggt cgatccgaac 1800

cgtcaggtgg cgattgccaa cccgatggcg ctgccgttcg tctctaagct tattccacgc 1860

ggtccgggca acccgatgga acagccgaaa gatgcaaaag gcacaggcac cgaatccggc 1920

atccagccgc agtacggtgt accgtatggc gtcacgctca atccgttcct ctcaccgttt 1980

ggtctgccat gtaaacagcc agcatggggt tatatttcgg cgctggatct gaaaaccaat 2040

gaagtggtgt ggaagaaacg cattggtacg ccgcaggaca gcatgccgtt cccgatgccg 2100

gttccgcttc ccttcaacat ggggatgccg atgctcggcg ggcccatctc gactgccggt 2160

aacgtgctgt ttatcgccgc tacggcagat aactacctgc gcgcttacaa catgagcaac 2220

ggtgaaaaac tgtggcaggg tcgtctacca gcgggcggtc aggcaacacc gatgacctat 2280

gaggtgaacg gcaagcagta tgtcgtgatt tcagccgggg gccacggctc gtttggtacg 2340

aagatgggcg attatattgt cgcgtatgcg ctgccggatg atgtgaaata a 2391

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<213> Artificial Sequence (Artificial Sequence)

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Met Ala Ile Asn Asn Thr Gly Ser Arg Arg Leu Leu Val Val Leu Thr

1 5 10 15

Ala Leu Phe Ala Ala Leu Cys Gly Leu Tyr Leu Leu Ile Gly Gly Gly

20 25 30

Trp Leu Val Ala Ile Gly Gly Ser Trp Tyr Tyr Pro Ile Ala Gly Leu

35 40 45

Ala Met Leu Gly Val Ala Trp Leu Leu Trp Arg Ser Lys Arg Ser Ala

50 55 60

Leu Trp Leu Tyr Ala Ala Leu Leu Leu Ala Thr Leu Ile Trp Gly Val

65 70 75 80

Trp Glu Val Gly Phe Asp Phe Trp Ala Leu Thr Pro Arg Ser Asp Ile

85 90 95

Leu Val Phe Phe Gly Ile Trp Leu Ile Leu Pro Phe Val Trp Arg Arg

100 105 110

Leu Val Ile Pro Ala Ser Gly Ala Val Ala Ala Leu Val Val Ala Leu

115 120 125

Leu Ile Ser Gly Gly Ile Leu Thr Trp Ala Gly Phe Asn Asp Pro Gln

130 135 140

Glu Ile Asp Gly Ala Leu Ser Ala Glu Ser Thr Pro Ala Gln Ala Ile

145 150 155 160

Ser Pro Val Ala Asp Gly Asp Trp Pro Ala Tyr Gly Arg Asn Gln Glu

165 170 175

Gly Gln Arg Phe Ser Pro Leu Lys Gln Ile His Ala Asp Asn Val His

180 185 190

Lys Leu Lys Glu Ala Trp Val Phe Arg Thr Gly Asp Val Lys Gln Pro

195 200 205

Asn Asp Pro Gly Glu Ile Thr Asn Glu Val Thr Pro Ile Lys Val Gly

210 215 220

Asp Thr Leu Tyr Leu Cys Thr Ala His Gln Arg Leu Phe Ala Leu Glu

225 230 235 240

Ala Ala Thr Gly Lys Glu Lys Trp His Tyr Asp Pro Glu Leu Lys Thr

245 250 255

Asn Glu Ser Phe Gln His Val Thr Cys Arg Gly Val Ser Tyr His Glu

260 265 270

Ala Lys Ala Glu Thr Ala Ser Pro Glu Val Met Ala Asp Cys Pro Arg

275 280 285

Arg Ile Ile Leu Pro Val Asn Asp Gly Arg Leu Ile Ala Ile Asn Ala

290 295 300

Glu Asn Gly Lys Leu Cys Glu Thr Phe Ala Asn Lys Gly Val Leu Asn

305 310 315 320

Leu Gln Ser Asn Met Pro Asp Thr Lys Pro Gly Leu Tyr Glu Pro Thr

325 330 335

Ser Pro Pro Ile Ile Thr Asp Lys Thr Ile Val Ile Ala Gly Ser Val

340 345 350

Thr Asp Asn Phe Ser Thr Arg Glu Thr Ser Gly Val Ile Arg Gly Phe

355 360 365

Asp Val Asn Asn Gly Lys Leu Leu Trp Ala Phe Asp Pro Gly Ala Lys

370 375 380

Asp Pro Asn Ala Ile Pro Ser Asp Glu His Ser Phe Thr Phe Asn Ser

385 390 395 400

Pro Asn Ser Trp Ala Pro Ala Ala Tyr Asp Ala Lys Leu Asp Leu Val

405 410 415

Tyr Leu Pro Met Gly Val Ser Thr Pro Asp Ile Trp Gly Gly His Arg

420 425 430

Thr Pro Glu Gln Glu Arg Tyr Ala Ser Ser Ile Leu Ala Leu Asn Ala

435 440 445

Thr Thr Gly Lys Leu Ala Trp Ser Tyr Gln Thr Val His His Asp Leu

450 455 460

Trp Asp Met Asp Met Pro Ser Gln Pro Thr Leu Ala Asp Ile Thr Val

465 470 475 480

Asn Gly Glu Lys Val Pro Val Ile Tyr Ala Pro Ala Lys Thr Gly Asn

485 490 495

Ile Phe Val Leu Asp Arg Arg Asn Gly Glu Leu Val Val Pro Ala Pro

500 505 510

Glu Lys Pro Val Pro Gln Gly Ala Ala Lys Gly Asp Tyr Val Thr Pro

515 520 525

Thr Gln Pro Phe Ser Glu Leu Ser Phe Arg Pro Thr Lys Asp Leu Ser

530 535 540

Gly Ala Asp Met Trp Gly Ala Thr Met Phe Asp Gln Leu Val Cys Arg

545 550 555 560

Val Met Phe His Gln Met Arg Tyr Glu Gly Ile Phe Thr Pro Pro Ser

565 570 575

Glu Gln Gly Thr Leu Val Phe Pro Gly Asn Leu Gly Met Phe Glu Trp

580 585 590

Gly Gly Ile Ser Val Asp Pro Asn Arg Gln Val Ala Ile Ala Asn Pro

595 600 605

Met Ala Leu Pro Phe Val Ser Lys Leu Ile Pro Arg Gly Pro Gly Asn

610 615 620

Pro Met Glu Gln Pro Lys Asp Ala Lys Gly Thr Gly Thr Glu Ser Gly

625 630 635 640

Ile Gln Pro Gln Tyr Gly Val Pro Tyr Gly Val Thr Leu Asn Pro Phe

645 650 655

Leu Ser Pro Phe Gly Leu Pro Cys Lys Gln Pro Ala Trp Gly Tyr Ile

660 665 670

Ser Ala Leu Asp Leu Lys Thr Asn Glu Val Val Trp Lys Lys Arg Ile

675 680 685

Gly Thr Pro Gln Asp Ser Met Pro Phe Pro Met Pro Val Pro Leu Pro

690 695 700

Phe Asn Met Gly Met Pro Met Leu Gly Gly Pro Ile Ser Thr Ala Gly

705 710 715 720

Asn Val Leu Phe Ile Ala Ala Thr Ala Asp Asn Tyr Leu Arg Ala Tyr

725 730 735

Asn Met Ser Asn Gly Glu Lys Leu Trp Gln Gly Arg Leu Pro Ala Gly

740 745 750

Gly Gln Ala Thr Pro Met Thr Tyr Glu Val Asn Gly Lys Gln Tyr Val

755 760 765

Val Ile Ser Ala Gly Gly His Gly Ser Phe Gly Thr Lys Met Gly Asp

770 775 780

Tyr Ile Val Ala Tyr Ala Leu Pro Asp Asp Val Lys

785 790 795

<210> 3

<211> 2391

<212> DNA

<213> Citrobacter rodentium

<400> 3

atggctgaaa acaatgcacg ttcgccacga cttctcgtga cgctgacggc cctctttgca 60

gcgctttgcg ggctgtatct tctgatcggc ggtggctggc tggtcgccat cggcggctcc 120

tggtactacc cgatcgccgg tctggcgatg ctgggcgtcg cctggctgct gtggcgcagc 180

agacgtacgg cgctatggct gtatgccgcc ctgctcctcg ccaccatgat ctggggcgta 240

tgggaagtcg gcttcgactt ctgggcgctg acgccgcgca gcgatatcct ggtcttcttc 300

ggcatctggc tgattttgcc ttttgtctgg catcgcctga tggtgccttc ccgcggcgcg 360

gtggccgcac tggttgccgc cctgctgatt agcggcggca tcctgacctg ggcgggcttc 420

aacgacccgc aggagatcga cggcgcgctc agcgcggagt cgacgcctgc acaggccatc 480

tcaccagtgg ctgacggcga ctggccggcg tatggccgca atcaggaagg ccagcgctat 540

tcgccgctga agcaaattaa cgccgataac gttcacaagc tgaaagaagc atgggtgttc 600

cgtaccggcg atctgaagca gccggacgat ccgggcgaac tgaccaatga agtgacgcca 660

attaaagtgg gcgacacgct gtatctgtgc accgctcacc agcgtctgtt cgcgctggag 720

gcggcgacgg gtaaagaaaa atggcactac gacccggagc tgaaaaccaa cgagtccttc 780

cagcacgtta cctgccgcgg cgtttcatac catgaggcga ctgcgggtaa cgcttcgccg 840

gaagtgattg ccgactgccc gcgccgcatt attctgccgg taaacgacgg tcgtctgatt 900

gcgcttaacg ctgaaaccgg caagctgtgc gagactttcg gcaacaaagg cgtgctcaat 960

ctgcaaacca acatgccgga tcaaacgccg gggctgtatg agccaacctc gccgccgatc 1020

atcaccgata aaaccatcgt cattgccggt tcggtgaccg ataacttctc gacccgcgag 1080

acttccggcg tcattcgcgg cttcgatgtt aacaacggca agctgctgtg ggcgttcgat 1140

ccgggcgcga aagacccgaa tgcgatcccg tccgatgagc acacgtttac ctttaactcg 1200

ccgaactcct gggcgccagc ggcctatgac gcgaagctgg acctcgttta cctgccgatg 1260

ggggtctcga cgccggatat ctggggcgga caccggacgc cggagcagga gcgctacgcc 1320

agttccattc tggcgctgaa cgcgaccacc ggtaaactcg cctggagcta tcagacggtt 1380

caccacgatc tgtgggatat ggacctgccc gctcagccga cgctggcgga cattaccgtc 1440

aacggccaga ccgttccggt catttacgcc ccggcgaaaa ccggcaatat ctttgtgctg 1500

gatcgccgta acggcgaact ggtggtgcct gcgccggaaa cgccggtgcc gcagggcgcc 1560

gcgaaaggcg attacgtcag caaaacgcag ccgttctctg aactgagctt ccgtccgaag 1620

aaagatctca gcggcgcgga tatgtggggc gccaccatgt tcgaccagct ggtatgccgc 1680

gtgatgttcc accagctgcg ctatgaaggc atcttcactc cgccatctga gcagggcacg 1740

ctggtgttcc cgggcaacct cgggatgttc gaatggggcg gtatttccgt cgatccgaac 1800

cgtcaggtag cgattgctaa cccgatggcg ctgccgttcg tctctaagct tattccacgc 1860

ggtccgggca acccgatgga gcagccgaag gatgcgaaag gcaccggcac cgaagccggt 1920

attcagccgc agtacggcgt accgtacggc gtgacgctga acccgttcct gtcgccgttt 1980

ggcctgccgt gtaagcaacc ggcctggggt tatatttccg cgctggatct gaaaaccaat 2040

gaagtggtgt ggaaaaaacg tatcggtacg ccgcaggaca gtatgccgtt cccgatgccg 2100

gttccgcttc ccttcaacat ggggatgccg atgctcggcg ggcccatctc gactgccggt 2160

aacgtgctgt ttatcgccgc gaccgccgat aactacctgc gcgcttacaa catgagcaac 2220

ggggaaaagc tgtggcaggc tcgcctgcca gcgggcggac aggccacgcc gatgacctat 2280

gaggtgaatg gcaagcagta cgttgttatt tccgcgggtg gtcacggttc gtttggtacg 2340

aagatgggcg attatattgt cgcgtatgcg ctgccggacg acgagaagta a 2391

<210> 4

<211> 796

<212> PRT

<213> Citrobacter rodentium

<400> 4

Met Ala Glu Asn Asn Ala Arg Ser Pro Arg Leu Leu Val Thr Leu Thr

1 5 10 15

Ala Leu Phe Ala Ala Leu Cys Gly Leu Tyr Leu Leu Ile Gly Gly Gly

20 25 30

Trp Leu Val Ala Ile Gly Gly Ser Trp Tyr Tyr Pro Ile Ala Gly Leu

35 40 45

Ala Met Leu Gly Val Ala Trp Leu Leu Trp Arg Ser Arg Arg Thr Ala

50 55 60

Leu Trp Leu Tyr Ala Ala Leu Leu Leu Ala Thr Met Ile Trp Gly Val

65 70 75 80

Trp Glu Val Gly Phe Asp Phe Trp Ala Leu Thr Pro Arg Ser Asp Ile

85 90 95

Leu Val Phe Phe Gly Ile Trp Leu Ile Leu Pro Phe Val Trp His Arg

100 105 110

Leu Met Val Pro Ser Arg Gly Ala Val Ala Ala Leu Val Ala Ala Leu

115 120 125

Leu Ile Ser Gly Gly Ile Leu Thr Trp Ala Gly Phe Asn Asp Pro Gln

130 135 140

Glu Ile Asp Gly Ala Leu Ser Ala Glu Ser Thr Pro Ala Gln Ala Ile

145 150 155 160

Ser Pro Val Ala Asp Gly Asp Trp Pro Ala Tyr Gly Arg Asn Gln Glu

165 170 175

Gly Gln Arg Tyr Ser Pro Leu Lys Gln Ile Asn Ala Asp Asn Val His

180 185 190

Lys Leu Lys Glu Ala Trp Val Phe Arg Thr Gly Asp Leu Lys Gln Pro

195 200 205

Asp Asp Pro Gly Glu Leu Thr Asn Glu Val Thr Pro Ile Lys Val Gly

210 215 220

Asp Thr Leu Tyr Leu Cys Thr Ala His Gln Arg Leu Phe Ala Leu Glu

225 230 235 240

Ala Ala Thr Gly Lys Glu Lys Trp His Tyr Asp Pro Glu Leu Lys Thr

245 250 255

Asn Glu Ser Phe Gln His Val Thr Cys Arg Gly Val Ser Tyr His Glu

260 265 270

Ala Thr Ala Gly Asn Ala Ser Pro Glu Val Ile Ala Asp Cys Pro Arg

275 280 285

Arg Ile Ile Leu Pro Val Asn Asp Gly Arg Leu Ile Ala Leu Asn Ala

290 295 300

Glu Thr Gly Lys Leu Cys Glu Thr Phe Gly Asn Lys Gly Val Leu Asn

305 310 315 320

Leu Gln Thr Asn Met Pro Asp Gln Thr Pro Gly Leu Tyr Glu Pro Thr

325 330 335

Ser Pro Pro Ile Ile Thr Asp Lys Thr Ile Val Ile Ala Gly Ser Val

340 345 350

Thr Asp Asn Phe Ser Thr Arg Glu Thr Ser Gly Val Ile Arg Gly Phe

355 360 365

Asp Val Asn Asn Gly Lys Leu Leu Trp Ala Phe Asp Pro Gly Ala Lys

370 375 380

Asp Pro Asn Ala Ile Pro Ser Asp Glu His Thr Phe Thr Phe Asn Ser

385 390 395 400

Pro Asn Ser Trp Ala Pro Ala Ala Tyr Asp Ala Lys Leu Asp Leu Val

405 410 415

Tyr Leu Pro Met Gly Val Ser Thr Pro Asp Ile Trp Gly Gly His Arg

420 425 430

Thr Pro Glu Gln Glu Arg Tyr Ala Ser Ser Ile Leu Ala Leu Asn Ala

435 440 445

Thr Thr Gly Lys Leu Ala Trp Ser Tyr Gln Thr Val His His Asp Leu

450 455 460

Trp Asp Met Asp Leu Pro Ala Gln Pro Thr Leu Ala Asp Ile Thr Val

465 470 475 480

Asn Gly Gln Thr Val Pro Val Ile Tyr Ala Pro Ala Lys Thr Gly Asn

485 490 495

Ile Phe Val Leu Asp Arg Arg Asn Gly Glu Leu Val Val Pro Ala Pro

500 505 510

Glu Thr Pro Val Pro Gln Gly Ala Ala Lys Gly Asp Tyr Val Ser Lys

515 520 525

Thr Gln Pro Phe Ser Glu Leu Ser Phe Arg Pro Lys Lys Asp Leu Ser

530 535 540

Gly Ala Asp Met Trp Gly Ala Thr Met Phe Asp Gln Leu Val Cys Arg

545 550 555 560

Val Met Phe His Gln Leu Arg Tyr Glu Gly Ile Phe Thr Pro Pro Ser

565 570 575

Glu Gln Gly Thr Leu Val Phe Pro Gly Asn Leu Gly Met Phe Glu Trp

580 585 590

Gly Gly Ile Ser Val Asp Pro Asn Arg Gln Val Ala Ile Ala Asn Pro

595 600 605

Met Ala Leu Pro Phe Val Ser Lys Leu Ile Pro Arg Gly Pro Gly Asn

610 615 620

Pro Met Glu Gln Pro Lys Asp Ala Lys Gly Thr Gly Thr Glu Ala Gly

625 630 635 640

Ile Gln Pro Gln Tyr Gly Val Pro Tyr Gly Val Thr Leu Asn Pro Phe

645 650 655

Leu Ser Pro Phe Gly Leu Pro Cys Lys Gln Pro Ala Trp Gly Tyr Ile

660 665 670

Ser Ala Leu Asp Leu Lys Thr Asn Glu Val Val Trp Lys Lys Arg Ile

675 680 685

Gly Thr Pro Gln Asp Ser Met Pro Phe Pro Met Pro Val Pro Leu Pro

690 695 700

Phe Asn Met Gly Met Pro Met Leu Gly Gly Pro Ile Ser Thr Ala Gly

705 710 715 720

Asn Val Leu Phe Ile Ala Ala Thr Ala Asp Asn Tyr Leu Arg Ala Tyr

725 730 735

Asn Met Ser Asn Gly Glu Lys Leu Trp Gln Ala Arg Leu Pro Ala Gly

740 745 750

Gly Gln Ala Thr Pro Met Thr Tyr Glu Val Asn Gly Lys Gln Tyr Val

755 760 765

Val Ile Ser Ala Gly Gly His Gly Ser Phe Gly Thr Lys Met Gly Asp

770 775 780

Tyr Ile Val Ala Tyr Ala Leu Pro Asp Asp Glu Lys

785 790 795

<210> 5

<211> 2454

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60

atgatggcta ttaacaatac aggctcgcga cgactcctcg tggtgttaac ggccctcttt 120

gcagctcttt gcgggctgta tcttctcatt ggcggaggct ggttggtcgc cattggcggc 180

tcctggtact acccgatcgc cggtctggcg atgctgggcg tcgcctggct gctgtggcgc 240

agcaaacgtt ccgcactctg gctgtacgcc gccctgctcc tcgccaccct gatttggggc 300

gtgtgggaag ttggtttcga cttctgggcg ctgactccgc gcagcgacat tctggtcttc 360

ttcggcatct ggctgatcct gccgtttgtc tggcgtcgcc tggtcattcc tgccagcggc 420

gcagttgccg cactggtggt cgcgctgttg attagcggtg gtatcctcac ctgggcgggc 480

ttcaacgacc cgcaggagat cgacggcgcg ctcagcgcgg agtcgacgcc tgcacaggcc 540

atctcaccag tggctgacgg cgactggccg gcgtatggcc gcaatcagga aggtcaacgc 600

ttttcaccac tgaagcaaat tcacgccgat aacgtccaca agctgaaaga agcctgggtg 660

ttccgtactg gcgatgtgaa gcagccgaac gatccgggtg aaatcaccaa tgaagtgacg 720

ccaattaaag tgggcgacac gctgtatctg tgcaccgctc accagcgtct gttcgcgctg 780

gaggcggcga cgggtaaaga aaaatggcat tacgatcctg agctgaaaac caacgagtct 840

ttccagcatg taacctgccg tggtgtctct tatcatgaag ccaaagcaga aactgcttcg 900

ccggaagtga tggcggattg cccgcgtcgt atcattctcc cggtcaatga tggccgcctg 960

attgcgatta acgctgaaaa cggcaagctg tgcgaaacct tcgctaataa aggcgtgctc 1020

aatctgcaaa gcaatatgcc agacaccaaa ccgggtctgt atgagccgac ttcgccgccg 1080

attatcaccg ataaaacgat tgtgattgct ggttcagtaa cggataactt ctccacccgc 1140

gaaacctcgg gcgtgatccg tggttttgac gtcaataacg gtaaactgct gtgggcgttc 1200

gatccgggtg cgaaagaccc gaatgcaatc ccttccgatg agcactcttt tacctttaac 1260

tcgccgaact cctgggcgcc agcggcctat gacgcgaagc tggacctcgt ttacctgccg 1320

atgggggtct cgacgccgga tatctggggc ggacaccgga cgccggagca ggagcgctac 1380

gccagttcca ttctggcgct gaacgcgacc accggtaaac tcgcctggag ctatcagacg 1440

gttcaccacg atctgtggga tatggacatg ccgtcccagc cgacgctggc ggatattacc 1500

gtcaacggtg agaaagtccc ggttatctac gcgccagcga aaaccggtaa catctttgtc 1560

ctcgaccgcc gtaacggcga gctggtcgtt cctgcaccgg aaaaaccggt tccgcagggg 1620

gccgcgaaag gcgattacgt tacccctact caaccgttct ctgagctgag cttccgtccg 1680

acaaaagatc taagcggtgc ggatatgtgg ggtgccacca tgtttgacca actggtgtgc 1740

cgcgtgatgt tccaccagat gcgctatgaa ggcattttca ccccaccatc tgaacagggt 1800

acgctggtct tcccgggtaa cctggggatg ttcgaatggg gcggtatttc ggtcgatccg 1860

aaccgtcagg tggcgattgc caacccgatg gcgctgccgt tcgtctctaa gcttattcca 1920

cgcggtccgg gcaacccgat ggaacagccg aaagatgcaa aaggcacagg caccgaatcc 1980

ggcatccagc cgcagtacgg tgtaccgtat ggcgtcacgc tcaatccgtt cctctcaccg 2040

tttggtctgc catgtaaaca gccagcatgg ggttatattt cggcgctgga tctgaaaacc 2100

aatgaagtgg tgtggaagaa acgcattggt acgccgcagg acagcatgcc gttcccgatg 2160

ccggttccgc ttcccttcaa catggggatg ccgatgctcg gcgggcccat ctcgactgcc 2220

ggtaacgtgc tgtttatcgc cgctacggca gataactacc tgcgcgctta caacatgagc 2280

aacggtgaaa aactgtggca gggtcgtcta ccagcgggcg gtcaggcaac accgatgacc 2340

tatgaggtga acggcaagca gtatgtcgtg atttcagccg ggggccacgg ctcgtttggt 2400

acgaagatgg gcgattatat tgtcgcgtat gcgctgccgg atgatgtgaa ataa 2454

<210> 6

<211> 817

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Met Ala Ile Asn Asn Thr Gly Ser Arg Arg Leu

20 25 30

Leu Val Val Leu Thr Ala Leu Phe Ala Ala Leu Cys Gly Leu Tyr Leu

35 40 45

Leu Ile Gly Gly Gly Trp Leu Val Ala Ile Gly Gly Ser Trp Tyr Tyr

50 55 60

Pro Ile Ala Gly Leu Ala Met Leu Gly Val Ala Trp Leu Leu Trp Arg

65 70 75 80

Ser Lys Arg Ser Ala Leu Trp Leu Tyr Ala Ala Leu Leu Leu Ala Thr

85 90 95

Leu Ile Trp Gly Val Trp Glu Val Gly Phe Asp Phe Trp Ala Leu Thr

100 105 110

Pro Arg Ser Asp Ile Leu Val Phe Phe Gly Ile Trp Leu Ile Leu Pro

115 120 125

Phe Val Trp Arg Arg Leu Val Ile Pro Ala Ser Gly Ala Val Ala Ala

130 135 140

Leu Val Val Ala Leu Leu Ile Ser Gly Gly Ile Leu Thr Trp Ala Gly

145 150 155 160

Phe Asn Asp Pro Gln Glu Ile Asp Gly Ala Leu Ser Ala Glu Ser Thr

165 170 175

Pro Ala Gln Ala Ile Ser Pro Val Ala Asp Gly Asp Trp Pro Ala Tyr

180 185 190

Gly Arg Asn Gln Glu Gly Gln Arg Phe Ser Pro Leu Lys Gln Ile His

195 200 205

Ala Asp Asn Val His Lys Leu Lys Glu Ala Trp Val Phe Arg Thr Gly

210 215 220

Asp Val Lys Gln Pro Asn Asp Pro Gly Glu Ile Thr Asn Glu Val Thr

225 230 235 240

Pro Ile Lys Val Gly Asp Thr Leu Tyr Leu Cys Thr Ala His Gln Arg

245 250 255

Leu Phe Ala Leu Glu Ala Ala Thr Gly Lys Glu Lys Trp His Tyr Asp

260 265 270

Pro Glu Leu Lys Thr Asn Glu Ser Phe Gln His Val Thr Cys Arg Gly

275 280 285

Val Ser Tyr His Glu Ala Lys Ala Glu Thr Ala Ser Pro Glu Val Met

290 295 300

Ala Asp Cys Pro Arg Arg Ile Ile Leu Pro Val Asn Asp Gly Arg Leu

305 310 315 320

Ile Ala Ile Asn Ala Glu Asn Gly Lys Leu Cys Glu Thr Phe Ala Asn

325 330 335

Lys Gly Val Leu Asn Leu Gln Ser Asn Met Pro Asp Thr Lys Pro Gly

340 345 350

Leu Tyr Glu Pro Thr Ser Pro Pro Ile Ile Thr Asp Lys Thr Ile Val

355 360 365

Ile Ala Gly Ser Val Thr Asp Asn Phe Ser Thr Arg Glu Thr Ser Gly

370 375 380

Val Ile Arg Gly Phe Asp Val Asn Asn Gly Lys Leu Leu Trp Ala Phe

385 390 395 400

Asp Pro Gly Ala Lys Asp Pro Asn Ala Ile Pro Ser Asp Glu His Ser

405 410 415

Phe Thr Phe Asn Ser Pro Asn Ser Trp Ala Pro Ala Ala Tyr Asp Ala

420 425 430

Lys Leu Asp Leu Val Tyr Leu Pro Met Gly Val Ser Thr Pro Asp Ile

435 440 445

Trp Gly Gly His Arg Thr Pro Glu Gln Glu Arg Tyr Ala Ser Ser Ile

450 455 460

Leu Ala Leu Asn Ala Thr Thr Gly Lys Leu Ala Trp Ser Tyr Gln Thr

465 470 475 480

Val His His Asp Leu Trp Asp Met Asp Met Pro Ser Gln Pro Thr Leu

485 490 495

Ala Asp Ile Thr Val Asn Gly Glu Lys Val Pro Val Ile Tyr Ala Pro

500 505 510

Ala Lys Thr Gly Asn Ile Phe Val Leu Asp Arg Arg Asn Gly Glu Leu

515 520 525

Val Val Pro Ala Pro Glu Lys Pro Val Pro Gln Gly Ala Ala Lys Gly

530 535 540

Asp Tyr Val Thr Pro Thr Gln Pro Phe Ser Glu Leu Ser Phe Arg Pro

545 550 555 560

Thr Lys Asp Leu Ser Gly Ala Asp Met Trp Gly Ala Thr Met Phe Asp

565 570 575

Gln Leu Val Cys Arg Val Met Phe His Gln Met Arg Tyr Glu Gly Ile

580 585 590

Phe Thr Pro Pro Ser Glu Gln Gly Thr Leu Val Phe Pro Gly Asn Leu

595 600 605

Gly Met Phe Glu Trp Gly Gly Ile Ser Val Asp Pro Asn Arg Gln Val

610 615 620

Ala Ile Ala Asn Pro Met Ala Leu Pro Phe Val Ser Lys Leu Ile Pro

625 630 635 640

Arg Gly Pro Gly Asn Pro Met Glu Gln Pro Lys Asp Ala Lys Gly Thr

645 650 655

Gly Thr Glu Ser Gly Ile Gln Pro Gln Tyr Gly Val Pro Tyr Gly Val

660 665 670

Thr Leu Asn Pro Phe Leu Ser Pro Phe Gly Leu Pro Cys Lys Gln Pro

675 680 685

Ala Trp Gly Tyr Ile Ser Ala Leu Asp Leu Lys Thr Asn Glu Val Val

690 695 700

Trp Lys Lys Arg Ile Gly Thr Pro Gln Asp Ser Met Pro Phe Pro Met

705 710 715 720

Pro Val Pro Leu Pro Phe Asn Met Gly Met Pro Met Leu Gly Gly Pro

725 730 735

Ile Ser Thr Ala Gly Asn Val Leu Phe Ile Ala Ala Thr Ala Asp Asn

740 745 750

Tyr Leu Arg Ala Tyr Asn Met Ser Asn Gly Glu Lys Leu Trp Gln Gly

755 760 765

Arg Leu Pro Ala Gly Gly Gln Ala Thr Pro Met Thr Tyr Glu Val Asn

770 775 780

Gly Lys Gln Tyr Val Val Ile Ser Ala Gly Gly His Gly Ser Phe Gly

785 790 795 800

Thr Lys Met Gly Asp Tyr Ile Val Ala Tyr Ala Leu Pro Asp Asp Val

805 810 815

Lys

<210> 7

<211> 2454

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60

atgatggctg aaaacaatgc acgttcgcca cgacttctcg tgacgctgac ggccctcttt 120

gcagcgcttt gcgggctgta tcttctgatc ggcggtggct ggctggtcgc catcggcggc 180

tcctggtact acccgatcgc cggtctggcg atgctgggcg tcgcctggct gctgtggcgc 240

agcagacgta cggcgctatg gctgtatgcc gccctgctcc tcgccaccat gatctggggc 300

gtatgggaag tcggcttcga cttctgggcg ctgacgccgc gcagcgatat cctggtcttc 360

ttcggcatct ggctgatttt gccttttgtc tggcatcgcc tgatggtgcc ttcccgcggc 420

gcggtggccg cactggttgc cgccctgctg attagcggcg gcatcctgac ctgggcgggc 480

ttcaacgacc cgcaggagat cgacggcgcg ctcagcgcgg agtcgacgcc tgcacaggcc 540

atctcaccag tggctgacgg cgactggccg gcgtatggcc gcaatcagga aggccagcgc 600

tattcgccgc tgaagcaaat taacgccgat aacgttcaca agctgaaaga agcatgggtg 660

ttccgtaccg gcgatctgaa gcagccggac gatccgggcg aactgaccaa tgaagtgacg 720

ccaattaaag tgggcgacac gctgtatctg tgcaccgctc accagcgtct gttcgcgctg 780

gaggcggcga cgggtaaaga aaaatggcac tacgacccgg agctgaaaac caacgagtcc 840

ttccagcacg ttacctgccg cggcgtttca taccatgagg cgactgcggg taacgcttcg 900

ccggaagtga ttgccgactg cccgcgccgc attattctgc cggtaaacga cggtcgtctg 960

attgcgctta acgctgaaac cggcaagctg tgcgagactt tcggcaacaa aggcgtgctc 1020

aatctgcaaa ccaacatgcc ggatcaaacg ccggggctgt atgagccaac ctcgccgccg 1080

atcatcaccg ataaaaccat cgtcattgcc ggttcggtga ccgataactt ctcgacccgc 1140

gagacttccg gcgtcattcg cggcttcgat gttaacaacg gcaagctgct gtgggcgttc 1200

gatccgggcg cgaaagaccc gaatgcgatc ccgtccgatg agcacacgtt tacctttaac 1260

tcgccgaact cctgggcgcc agcggcctat gacgcgaagc tggacctcgt ttacctgccg 1320

atgggggtct cgacgccgga tatctggggc ggacaccgga cgccggagca ggagcgctac 1380

gccagttcca ttctggcgct gaacgcgacc accggtaaac tcgcctggag ctatcagacg 1440

gttcaccacg atctgtggga tatggacctg cccgctcagc cgacgctggc ggacattacc 1500

gtcaacggcc agaccgttcc ggtcatttac gccccggcga aaaccggcaa tatctttgtg 1560

ctggatcgcc gtaacggcga actggtggtg cctgcgccgg aaacgccggt gccgcagggc 1620

gccgcgaaag gcgattacgt cagcaaaacg cagccgttct ctgaactgag cttccgtccg 1680

aagaaagatc tcagcggcgc ggatatgtgg ggcgccacca tgttcgacca gctggtatgc 1740

cgcgtgatgt tccaccagct gcgctatgaa ggcatcttca ctccgccatc tgagcagggc 1800

acgctggtgt tcccgggcaa cctcgggatg ttcgaatggg gcggtatttc cgtcgatccg 1860

aaccgtcagg tagcgattgc taacccgatg gcgctgccgt tcgtctctaa gcttattcca 1920

cgcggtccgg gcaacccgat ggagcagccg aaggatgcga aaggcaccgg caccgaagcc 1980

ggtattcagc cgcagtacgg cgtaccgtac ggcgtgacgc tgaacccgtt cctgtcgccg 2040

tttggcctgc cgtgtaagca accggcctgg ggttatattt ccgcgctgga tctgaaaacc 2100

aatgaagtgg tgtggaaaaa acgtatcggt acgccgcagg acagtatgcc gttcccgatg 2160

ccggttccgc ttcccttcaa catggggatg ccgatgctcg gcgggcccat ctcgactgcc 2220

ggtaacgtgc tgtttatcgc cgcgaccgcc gataactacc tgcgcgctta caacatgagc 2280

aacggggaaa agctgtggca ggctcgcctg ccagcgggcg gacaggccac gccgatgacc 2340

tatgaggtga atggcaagca gtacgttgtt atttccgcgg gtggtcacgg ttcgtttggt 2400

acgaagatgg gcgattatat tgtcgcgtat gcgctgccgg acgacgagaa gtaa 2454

<210> 8

<211> 817

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Met Ala Glu Asn Asn Ala Arg Ser Pro Arg Leu

20 25 30

Leu Val Thr Leu Thr Ala Leu Phe Ala Ala Leu Cys Gly Leu Tyr Leu

35 40 45

Leu Ile Gly Gly Gly Trp Leu Val Ala Ile Gly Gly Ser Trp Tyr Tyr

50 55 60

Pro Ile Ala Gly Leu Ala Met Leu Gly Val Ala Trp Leu Leu Trp Arg

65 70 75 80

Ser Arg Arg Thr Ala Leu Trp Leu Tyr Ala Ala Leu Leu Leu Ala Thr

85 90 95

Met Ile Trp Gly Val Trp Glu Val Gly Phe Asp Phe Trp Ala Leu Thr

100 105 110

Pro Arg Ser Asp Ile Leu Val Phe Phe Gly Ile Trp Leu Ile Leu Pro

115 120 125

Phe Val Trp His Arg Leu Met Val Pro Ser Arg Gly Ala Val Ala Ala

130 135 140

Leu Val Ala Ala Leu Leu Ile Ser Gly Gly Ile Leu Thr Trp Ala Gly

145 150 155 160

Phe Asn Asp Pro Gln Glu Ile Asp Gly Ala Leu Ser Ala Glu Ser Thr

165 170 175

Pro Ala Gln Ala Ile Ser Pro Val Ala Asp Gly Asp Trp Pro Ala Tyr

180 185 190

Gly Arg Asn Gln Glu Gly Gln Arg Tyr Ser Pro Leu Lys Gln Ile Asn

195 200 205

Ala Asp Asn Val His Lys Leu Lys Glu Ala Trp Val Phe Arg Thr Gly

210 215 220

Asp Leu Lys Gln Pro Asp Asp Pro Gly Glu Leu Thr Asn Glu Val Thr

225 230 235 240

Pro Ile Lys Val Gly Asp Thr Leu Tyr Leu Cys Thr Ala His Gln Arg

245 250 255

Leu Phe Ala Leu Glu Ala Ala Thr Gly Lys Glu Lys Trp His Tyr Asp

260 265 270

Pro Glu Leu Lys Thr Asn Glu Ser Phe Gln His Val Thr Cys Arg Gly

275 280 285

Val Ser Tyr His Glu Ala Thr Ala Gly Asn Ala Ser Pro Glu Val Ile

290 295 300

Ala Asp Cys Pro Arg Arg Ile Ile Leu Pro Val Asn Asp Gly Arg Leu

305 310 315 320

Ile Ala Leu Asn Ala Glu Thr Gly Lys Leu Cys Glu Thr Phe Gly Asn

325 330 335

Lys Gly Val Leu Asn Leu Gln Thr Asn Met Pro Asp Gln Thr Pro Gly

340 345 350

Leu Tyr Glu Pro Thr Ser Pro Pro Ile Ile Thr Asp Lys Thr Ile Val

355 360 365

Ile Ala Gly Ser Val Thr Asp Asn Phe Ser Thr Arg Glu Thr Ser Gly

370 375 380

Val Ile Arg Gly Phe Asp Val Asn Asn Gly Lys Leu Leu Trp Ala Phe

385 390 395 400

Asp Pro Gly Ala Lys Asp Pro Asn Ala Ile Pro Ser Asp Glu His Thr

405 410 415

Phe Thr Phe Asn Ser Pro Asn Ser Trp Ala Pro Ala Ala Tyr Asp Ala

420 425 430

Lys Leu Asp Leu Val Tyr Leu Pro Met Gly Val Ser Thr Pro Asp Ile

435 440 445

Trp Gly Gly His Arg Thr Pro Glu Gln Glu Arg Tyr Ala Ser Ser Ile

450 455 460

Leu Ala Leu Asn Ala Thr Thr Gly Lys Leu Ala Trp Ser Tyr Gln Thr

465 470 475 480

Val His His Asp Leu Trp Asp Met Asp Leu Pro Ala Gln Pro Thr Leu

485 490 495

Ala Asp Ile Thr Val Asn Gly Gln Thr Val Pro Val Ile Tyr Ala Pro

500 505 510

Ala Lys Thr Gly Asn Ile Phe Val Leu Asp Arg Arg Asn Gly Glu Leu

515 520 525

Val Val Pro Ala Pro Glu Thr Pro Val Pro Gln Gly Ala Ala Lys Gly

530 535 540

Asp Tyr Val Ser Lys Thr Gln Pro Phe Ser Glu Leu Ser Phe Arg Pro

545 550 555 560

Lys Lys Asp Leu Ser Gly Ala Asp Met Trp Gly Ala Thr Met Phe Asp

565 570 575

Gln Leu Val Cys Arg Val Met Phe His Gln Leu Arg Tyr Glu Gly Ile

580 585 590

Phe Thr Pro Pro Ser Glu Gln Gly Thr Leu Val Phe Pro Gly Asn Leu

595 600 605

Gly Met Phe Glu Trp Gly Gly Ile Ser Val Asp Pro Asn Arg Gln Val

610 615 620

Ala Ile Ala Asn Pro Met Ala Leu Pro Phe Val Ser Lys Leu Ile Pro

625 630 635 640

Arg Gly Pro Gly Asn Pro Met Glu Gln Pro Lys Asp Ala Lys Gly Thr

645 650 655

Gly Thr Glu Ala Gly Ile Gln Pro Gln Tyr Gly Val Pro Tyr Gly Val

660 665 670

Thr Leu Asn Pro Phe Leu Ser Pro Phe Gly Leu Pro Cys Lys Gln Pro

675 680 685

Ala Trp Gly Tyr Ile Ser Ala Leu Asp Leu Lys Thr Asn Glu Val Val

690 695 700

Trp Lys Lys Arg Ile Gly Thr Pro Gln Asp Ser Met Pro Phe Pro Met

705 710 715 720

Pro Val Pro Leu Pro Phe Asn Met Gly Met Pro Met Leu Gly Gly Pro

725 730 735

Ile Ser Thr Ala Gly Asn Val Leu Phe Ile Ala Ala Thr Ala Asp Asn

740 745 750

Tyr Leu Arg Ala Tyr Asn Met Ser Asn Gly Glu Lys Leu Trp Gln Ala

755 760 765

Arg Leu Pro Ala Gly Gly Gln Ala Thr Pro Met Thr Tyr Glu Val Asn

770 775 780

Gly Lys Gln Tyr Val Val Ile Ser Ala Gly Gly His Gly Ser Phe Gly

785 790 795 800

Thr Lys Met Gly Asp Tyr Ile Val Ala Tyr Ala Leu Pro Asp Asp Glu

805 810 815

Lys

Claims (9)

1. A method for constructing a recombinant microorganism having a phosphorus solubilizing activity, characterized in that: the method comprises the steps of introducing a coding gene of glucose dehydrogenase into a recipient microorganism to obtain a recombinant microorganism with a phosphate solubilizing activity higher than that of the recipient microorganism; the glucose dehydrogenase is a protein of a) or b) or c):

a) a protein consisting of an amino acid sequence shown in SEQ ID No. 2;

b) a protein consisting of an amino acid sequence shown as SEQ ID No. 6;

c) a fusion protein obtained by fusing a histidine tag at the carboxyl terminal of the protein shown in a) or b).

2. The method of claim 1, wherein: the coding gene is a gene shown in the following 1) or 2):

1) the coding sequence is a DNA molecule shown in SEQ ID No. 1;

2) the coding sequence is a DNA molecule shown in SEQ ID No. 5.

3. The method according to claim 1 or 2, characterized in that: the recipient microorganism is a prokaryotic microorganism.

4. The method of claim 3, wherein: the prokaryotic microorganism is a gram-negative bacterium or a gram-positive bacterium.

5. The method of claim 4, wherein: the gram-negative bacterium is an escherichia bacterium, and the gram-positive bacterium is a bacillus bacterium.

6. Use of the process according to any one of claims 1 to 5 for dissolving inorganic phosphorus.

7. A biomaterial related to the glucose dehydrogenase as claimed in claim 1, which is B1) or B2):

B1) an expression cassette comprising the coding gene of claim 1 or 2;

B2) a recombinant vector comprising the coding gene of claim 1 or 2.

8. Use of the biomaterial of claim 7 in the preparation of glucose dehydrogenase.

9. Use of the biomaterial of claim 7 in the preparation of a recombinant microorganism having phosphate solubilizing activity.

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