CN109402084B

CN109402084B - Method for constructing recombinant microorganism with phosphorus-solubilizing activity and nucleic acid molecule used in method

Info

Publication number: CN109402084B
Application number: CN201811351550.2A
Authority: CN
Inventors: 孙静文; 周卫; 程明芳; 李书田; 王玉军
Original assignee: Institute of Agricultural Resources and Regional Planning of CAAS
Current assignee: Institute of Agricultural Resources and Regional Planning of CAAS
Priority date: 2016-01-26
Filing date: 2016-01-26
Publication date: 2021-09-07
Anticipated expiration: 2036-01-26
Also published as: CN109402084A; CN105779408A; CN105779408B; CN109234251B; CN109234251A

Abstract

The invention discloses a method for constructing a recombinant microorganism with phosphorus-solubilizing activity and a nucleic acid molecule used by the same. The method comprises the following steps of introducing coding genes of the following proteins into a receptor microorganism to obtain a recombinant microorganism with phosphorus solubilizing activity, wherein the phosphorus solubilizing activity of the recombinant microorganism is higher than that of the receptor microorganism: a) a protein consisting of the amino acid sequence shown in the 1 st to 203 rd positions of SEQ ID No. 2; b) a protein consisting of the amino acid sequence shown in the 26 th to 203 rd positions of SEQ ID No. 2; 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 acid phosphatase activity is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequences shown in SEQ ID No.2 or SEQ ID No. 6. The invention can be used for cultivating bioengineering bacteria for efficiently activating the phosphorus nutrient in soil.

Description

Method for constructing recombinant microorganism with phosphorus-solubilizing activity and nucleic acid molecule used in method

The application is a divisional application with the application number of 201610051877.2, the application date of 2016, 26/01, and the name of application of acid phosphatase and related biomaterials in construction of phosphate solubilizing engineering bacteria.

Technical Field

The invention relates to a method for constructing a recombinant microorganism with phosphorus-solubilizing activity in the field of biology and a nucleic acid molecule used by the same.

Background

Phosphorus is one of three essential elements for plant growth and development and plays an important role in the life process. The phosphorus utilized by plants is mainly derived from soil. At present, 2/3 cultivated land is lack of phosphorus in China, and the main reason of the lack of phosphorus is that the content of available phosphorus in soil is insufficient, about 95 percent of phosphorus is insoluble invalid phosphorus, and plants are difficult to absorb and utilize. 1050-1200 million tons of phosphate fertilizer are consumed in China every year (2010, China chemical information network), but the utilization rate of the current-season plant of the phosphate fertilizer is only 5% -25%, and about 90% of the phosphate fertilizer is quickly chemically fixed after being applied to soil. Therefore, the improvement of the utilization efficiency of the phosphate fertilizer and the activation of the ineffective phosphorus in the soil are one of the scientific problems which are urgently solved by agricultural production.

The decomposition and release of organic phosphorus in soil by acid phosphatase and phytase in phosphate-solubilizing microorganisms play a key role (Yamamura et al, 2004; Zhao Xiao Rong et al, 2001; Zhenji et al, 2009). Acid phosphatase (ACPase for short, E.C.3.1.3.2) is an enzyme that catalyzes the hydrolysis of a phosphoric monoester under acidic conditions. Besides being involved in phosphate metabolism, the enzyme is also involved in important vital activities such as metabolic regulation, energy conversion and signal transduction. The acid phosphatase has very important functions, and the acid phosphatase has the activity of phosphohydrolase, releases phosphorus by decomposing phosphorus-ester bonds and phosphorus-anhydride bonds of organic matters, so as to activate ineffective phosphorus in soil, and has important application value in fully utilizing soil phosphorus resources and reducing the application of phosphate fertilizers; secondly, the acid phosphatase also has phosphotransferase activity, can transfer low-energy phosphate groups to nucleoside hydroxyl under proper conditions, and has great application value in nucleotide biochemical synthesis. The nucleotide is generally used as a food additive and a medical intermediate, wherein inosinic acid (hypoxanthine-5' -nucleotide) has a more obvious fresh-keeping effect and is widely applied to the field of food processing. At present, there are two main methods for producing nucleotide, one is chemical synthesis method, which uses thallus to ferment and produce inosine, and uses POCl₃Phosphorylation, more by-products and difficult purification of the method; another method is to phosphorylate inosine using E.coli inosine kinase, which requires the participation of ATP, which is fermented by AminobacterRegeneration, limits the application of enzymatic synthesis.

Disclosure of Invention

The invention aims to solve the technical problem of providing acid phosphatase with high phosphohydrolase activity.

In order to solve the above technical problems, the present invention provides the use of the protein of a) or b) or c) or d) as a phosphohydrolase:

a) a protein consisting of the amino acid sequence shown in the 1 st to 203 rd positions of SEQ ID No. 2;

b) a protein consisting of the amino acid sequence shown in the 26 th to 203 rd positions of SEQ ID No. 2;

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

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

In the application, the protein shown in a) is complete acid phosphatase derived from Bacillus megaterium (Bacillus megaterium), and the name of the protein is BmacpA; SEQ ID No.2 consists of 203 amino acid residues, and positions 1-25 are signal peptides.

In the above applications, the protein represented by b) is a signal peptide-removed acid phosphatase obtained by removing a signal peptide of BmacpA, and is named NSBmacpA.

In the above application, 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. c) The protein can be specifically a fusion protein obtained by fusing a histidine tag at the carboxyl terminal or/and the amino terminal of NSBmacpA, such as the protein shown in SEQ ID No.6, and the name of the protein is NSBmacpA-His. SEQ ID No.6 consists of 192 amino acid residues.

In order to solve the above technical problems, the present invention provides the use of a nucleic acid molecule in the preparation of a phosphohydrolase; the nucleic acid molecule encodes a protein of a) or b) or c) or d) above.

In the above application, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The nucleic acid molecule may specifically be a gene represented by the following 1) or 2) or 3) or 4):

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

2) the coding sequence is a DNA molecule shown in the 76 th to 612 th positions of SEQ ID No.1 and is named as NSBmacpA gene;

3) the coding sequence is a DNA molecule shown in SEQ ID No. 5; its name is NSBmacpA-His gene;

4) a DNA molecule having 90% or more identity to the DNA molecule defined in 1) or 2) or 3) and encoding the above-mentioned acid phosphatase.

In the above application, "identity" refers to sequence similarity to a 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.

Another technical problem to be solved by the present invention is how to construct microorganisms having phosphorus solubilizing activity.

In order to solve the above technical problems, the present invention provides the use of the above nucleic acid molecule for constructing a microorganism having a phosphate solubilizing activity.

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

The method for constructing the recombinant microorganism with the phosphorus solubilizing activity provided by the invention comprises the step of introducing the coding gene of the protein of a) or b) or c) or d) into a receptor microorganism to obtain the recombinant microorganism with the phosphorus solubilizing activity higher than that of the receptor microorganism.

In the above method, the phosphorus solubilizing activity is expressed as phosphohydrolase activity.

In the above method, the coding gene is a DNA molecule represented by the following 1) or 2) or 3) or 4):

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

2) the coding sequence is a DNA molecule shown in the 76 th to 612 th positions of SEQ ID No. 1;

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

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

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

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

In the above method, the gram-negative bacterium may specifically be an Escherichia bacterium or a Citrobacter bacterium.

In the above method, the Escherichia bacterium may specifically be Escherichia coli, such as Escherichia coli BL21(DE 3). The Citrobacter bacteria may be Citrobacter ACCC 02187.

In order to solve the above technical problems, the present invention provides a biomaterial containing the nucleic acid molecule.

The biological material containing the nucleic acid molecules provided by the invention is B1), B2), B3) or B4):

B1) an expression cassette comprising the nucleic acid molecule;

B2) a recombinant vector comprising said nucleic acid molecule;

B3) a recombinant vector containing the expression cassette;

B4) the recombinant microorganism with the phosphorus-solubilizing activity constructed by any one of the methods.

The expression cassette containing the nucleic acid molecule in the above-mentioned biological material is a DNA capable of expressing the protein of the above-mentioned a) or b) or c) or d) in a host cell, and the DNA may contain not only a promoter for promoting the transcription of the above-mentioned protein gene but also a terminator for terminating the transcription of the above-mentioned protein gene. The recombinant vector may be pET-NSBmacpA or pHT-BmacpA. The pET-NSBmacpA is introduced into the recipient microorganism; the pET-NSBmacpA is a recombinant expression vector obtained by replacing a fragment between NdeI and HindIII recognition sites of pET-30b (+) by a DNA molecule shown by nucleotides from 4 th to 537 th in a sequence 5 in a sequence table; the pHT-BmacpA is a recombinant expression vector obtained by replacing a fragment between BamHI and XbaI recognition sites of pHT43 by a DNA molecule shown in a sequence 1 in a sequence table.

Experiments prove that BmacpA and NSBmacpA both have the phosphohydrolase activity, and the phosphohydrolase activity of BmacpA and NSBmacpA in 50mmol/L acetic acid-sodium acetate buffer solution with the temperature of 37 ℃ and the pH value of 5.0 is 33.96 +/-1.32U/ml protein and 37.35 +/-1.55U/ml protein respectively; the acid phosphatase engineering bacteria obtained by introducing BmacpA gene into Citrobacter were found to have increased effective phosphorus contents of 14.59. mu. mol/L, 16.63. mu. mol/L and 18.55. mu. mol/L in the nucleic acid broth (containing salmon sperm DNA), the phospholipid broth (containing L-A-phosphatidylinositol) and the phosphoinositide broth (containing 1,4, 5-triphosphate inositol), respectively, as compared with Citrobacter as the recipient bacteria (FIG. 7). The invention can be used for cultivating bioengineering bacteria for efficiently activating the phosphorus nutrient in soil.

Drawings

FIG. 1 is an SDS-PAGE pattern of the induction expression of acid phosphatase in E.coli.

In fig. 1, M: a protein Marker; 1: blank reference bacterium crude enzyme liquid; 2: the crude enzyme solution of the empty vector control bacteria; 3: crude enzyme solution of NSBmacpB-His; 4: crude enzyme solution of NSBmacpA-His.

FIG. 2 is a graph of the effect of different pH and reaction time on phosphotransferase activity of NSBmacpA-His.

FIG. 3 is a graph showing the effect of different pH and reaction time on phosphotransferase activity of NSBmacpB-His.

FIG. 4 is a graph of the effect of different divalent ions on NSBmacpA-His and NSBmacpB-His phosphohydrolase activity.

In FIG. 4, BmacpA is NSBmacpA-His; BmacpB is NSBmacpB-His.

FIG. 5 is the SDS-PAGE expression map of acid phosphatase in the recombinant engineering bacteria.

In fig. 5, M: a protein Marker; 1: intracellular supernatant; 2: extracellular supernatant.

FIG. 6 is a graph showing the effect of pH on acid phosphatase enzyme activity of acid phosphatase engineering bacteria.

FIG. 7 shows the phosphate solubilizing effect of the acid phosphatase engineering bacteria in the medium supplemented with an organic phosphate source.

In FIG. 7, Citrobacter is Citrobacter ACCC02187, and Citrobacter into which acid phosphatase has been transferred is an acid phosphatase 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.

Bacillus megaterium ACCC10010 (China invention patent with publication number CN1189086C, 2/16.2005) used in the following examples was collected at the agricultural microorganism center of China Committee for culture Collection of microorganisms (ACCC for short, address: No. 12 south street of Guancun, Haidian, Beijing, institute for agricultural resources and agricultural division, postal code 100081) before the filing date of this application, and the strain was available to the public from the agricultural microorganism center of China Committee for culture Collection of microorganisms. In the invention, the bacillus megaterium ACCC10010 is shortened.

Bacillus megaterium (ACCC 02970) used in the following examples was collected by the agricultural microorganism center of China Committee for culture Collection of microorganisms (ACCC for short, address: southern Avenue 12 of Guancun in Haizu, Beijing, institute for agricultural resources and agricultural division, postal code 100081) before the filing date of this application, and the collection date thereof was 12 months and 20 days in 2007, from which the strain was obtained by the public from the agricultural microorganism center of China Committee for culture Collection. The bacillus megaterium ACCC02970 is shortened in the invention.

The Citrobacter sp (ACCC 02187) was collected by the agricultural microorganism center of China Committee for culture Collection of microorganisms (ACCC for short, address: southern street 12 of Guancun, Haizhou, Beijing, academy of agricultural resources and agricultural division, postal code 100081) before the date of filing of the present application, and was collected at 11/1/2005, from the date of collection, the strain was available to the public from the agricultural microorganism center of China Committee for culture Collection of microorganisms. The invention is called citric acid bacillus ACCC02187 for short.

Example 1 preparation and functional verification of acid phosphatase BmacpA

Cloning of BmacpA Gene and BmacpB Gene

Respectively extracting genome DNA of bacillus megaterium ACCC10010 and Bacillus megaterium ACCC02970, and carrying out PCR amplification on BmacpA genes by taking the genome DNA of the bacillus megaterium ACCC10010 as a template and P1 and P2 as primers; the BmacpB gene is amplified by PCR by taking the genome DNA of the bacillus megaterium ACCC02970 as a template and P3 and P4 as primers. Wherein the sequences of P1, P2, P3 and P4 are as follows: p1: 5'-ATGTATGTGAAACGATATCG-3', P2: 5'-CTACTTTTGTCGAACACATA-3', P3: 5'-ATGGTAAATCGCACTACAAA-3', P4: 5'-CTATTTTTGGTTATATAAGC-3' are provided.

Respectively carrying out electrophoresis on the obtained BmacpA gene PCR product and the BmacpB gene PCR product, wherein the result shows that the BmacpA gene PCR product and the BmacpB gene PCR product are bands of about 600bp, respectively recovering the BmacpA gene PCR product and the BmacpB gene PCR product, respectively and independently connecting the BmacpA gene PCR product and the BmacpB gene PCR product to a cloning vector, screening and identifying positive clones, and carrying out sequence determination; the sequencing result shows that the DNA sequence of the PCR product of the BmacpA gene is shown as a sequence 1 in a sequence table, the sequence 1 in the sequence table consists of 612 nucleotides, the protein shown as a sequence 2 in the coding sequence table is coded, the sequence 2 in the sequence table consists of 203 amino acid residues, the protein shown as the sequence 2 in the sequence table is named as BmacpA, the 1 st to 25 th sites of the sequence 2 are signal peptide sequences, and the DNA molecule shown as the sequence 1 in the sequence table is the BmacpA gene; the DNA sequence of the PCR product of the BmacpB gene is shown as a sequence 3 in a sequence table, the sequence 3 in the sequence table consists of 627 nucleotides, the protein shown as a sequence 4 in the sequence table is coded, the sequence 4 in the sequence table consists of 208 amino acid residues, the protein shown as the sequence 4 in the sequence table is named as BmacpB, the 1 st to 25 th sites of the sequence 4 in the sequence table are signal peptide sequences, and the DNA molecule shown as the sequence 3 in the sequence table is the BmacpB gene.

Second, construction of recombinant expression vector

1. Construction of pET-NSBmacpA

Designing BmacpA gene expression primer with signal peptide sequence eliminated. According to Bacillus megaterium ACCC10010

A signal peptide primer is designed in the BmacpA gene functional structural domain, an NdeI restriction site (CATATG) is added to a primer at the 5 'end from which the signal peptide is removed, and a HindIII restriction site (AAGCTT) is added to a primer at the 3' end. The upstream and downstream primers are respectively: p5 primer, 5 ' -AT-CATATG-TTTAATACACCTTGGGTGAA-3 ' and P6 primer, 5 ' -GC-AAGCTT-CTTTTGT CGAACACATAA-3'. By utilizing a PCR amplification method, signal peptide coding DNA (nucleotides 1 to 75 of a sequence 1 in a sequence table) is removed from a BmacpA gene coding region, NdeI and HindIII enzyme recognition sites are respectively introduced into a 5 'end and a 3' end to obtain a BmacpA gene PCR product without a signal peptide, the BmacpA gene without the signal peptide is named as NSBmacpA gene, and the BmacpA gene PCR product without the signal peptide is named as NSBmacpA gene PCR product. The PCR product of the NSBmacpA gene contains nucleotides 76 to 609 in a sequence 1 in a sequence table (nucleotides 4 to 537 in a sequence 5 in the sequence table).

Digesting the NSBmacpA gene PCR product obtained in the first step by NdeI and HindIII, and recovering a target fragment (NSBmacpA gene); meanwhile, NdeI and HindIII are used for enzyme digestion of the vector pET-30b (+) (EMD Biosciences purchased from Beijing New York Co., Ltd.), and a large vector fragment is recovered; and connecting the recovered target fragment with the recovered vector large fragment at 16 ℃ to obtain the target plasmid. Using CaCl as the target plasmid₂The method transforms Escherichia coli DH5 alpha competent cells. The suspension was uniformly spread on an LB plate containing ampicillin, and cultured at 37 ℃ for 16 hours. And carrying out shake culture on a single colony overnight, extracting a plasmid, carrying out double enzyme digestion identification by using NdeI and HindIII, sequencing the plasmid with correct enzyme digestion verification, and replacing a fragment between NdeI and HindIII recognition sites of pET-30b (+) by using a DNA molecule shown in the 4 th to 537 th sites of a sequence 5 in a sequence table to obtain a recombinant expression vector named as pET-NSBmacPA. pET-NSBmacpA contains His tag fusion protein NSBmacpA-His coding gene, NSBmacThe nucleotide sequence of the pA-His coding gene is a sequence 5 in the sequence table, and the NSBmacpA-His is a protein shown as a sequence 6 in the sequence table.

2. Construction of pET-NSBmacpB

BmacpB gene expression primers were designed with the signal peptide sequence removed. A primer is designed according to a BmacpB gene coding region sequence of Bacillus megaterium ACCC02970, an NdeI restriction site (CATATG) is added to a 5 'end primer with a signal peptide removed, and a HindIII restriction site (AAGCTT) is added to a 3' end primer. The upstream and downstream primers are respectively: p7 primer, 5' -AT-CATATGThe primers-TTTAA TACACCTTGG GTGAA-3 'and P8, 5' -GC-AAGCTT-TTTTTGG TTATATAAGCG-3'. Removing signal peptide from the BmacpB gene coding region by using a PCR amplification method, introducing NdeI and HindIII recognition sites into the 5 'end and the 3' end respectively to obtain a BmacpB gene PCR product without the signal peptide, naming the BmacpB gene without the signal peptide as NSBmacpB gene, and naming the BmacpB gene PCR product without the signal peptide as NSBmacpB gene PCR product. The PCR product of the NSBmacpB gene contains nucleotides 76 to 624 of a sequence 3 in a sequence table (nucleotides 4 to 552 of a sequence 7 in the sequence table).

Digesting the NSBmacpB gene PCR product obtained in the first step by NdeI and HindIII, and recovering a target fragment; meanwhile, NdeI and HindIII are used for enzyme digestion of the vector pET-30b (+) (EMD Biosciences purchased from Beijing New York Co., Ltd.), and a large vector fragment is recovered; and connecting the recovered target fragment with the recovered vector large fragment at 16 ℃ to obtain the target plasmid. Using CaCl as the target plasmid₂The method transforms Escherichia coli DH5 alpha competent cells. The suspension was uniformly spread on an LB plate containing ampicillin, and cultured at 37 ℃ for 16 hours. The single colony is shake cultured overnight, the extracted plasmid is double-enzyme-cut identified by NdeI and HindIII, the plasmid with correct enzyme-cut verification is sequenced, and the sequencing result shows that the DNA molecule shown by the 4 th to 552 th nucleotides in the sequence 7 in the sequence table is used for replacing the segment between the NdeI and HindIII recognition sites of pET-30b to obtain the recombinant expression vector which is named as pET-NSBmacpB. pET-NSBmacpB contains His label fusion protein NSBmacpB-His coding gene, the nucleotide sequence of the NSBmacpB-His coding gene is shown as sequence 7 in the sequence table, and NSBmacpB-His is shown as sequence 8 in the sequence tableA protein.

Preparation of recombinant Escherichia coli expressing acid phosphatase

1. Expression of NSBmacpA-His

And (3) transforming the pET-NSBmacpA in the second step into escherichia coli BL21(DE3) by a calcium chloride method (Tiangen corporation), screening and culturing positive clones by kanamycin resistance screening, selecting single clones, carrying out PCR identification by taking the P5 and the P6 as primers, and taking the positive clones which are PCR products of which the PCR products are determined to be about 539bp as genetically engineered bacteria and named as pET-NSBmacpA/BL 21. The pET-NSBmacpA/BL 21 strain was selected, inoculated into LB medium containing 100ug/ml kanamycin (a medium obtained by adding kanamycin to LB medium to 100ug/ml kanamycin concentration), and cultured at 37 ℃ to 0D₆₀₀When the value (LB medium containing 100ug/ml kanamycin as blank control) reached 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 resuspending the thallus with 50mM Tris-HCl (pH7.1) to obtain thallus content of 10⁸cfu/ml thallus suspension is subjected to ultrasonic disruption, is centrifuged at 12000 r/min for 10min, and supernatant (containing total thallus protein) is collected and named as NSBmacpA-His crude enzyme solution.

2. Expression of NSBmacpB-His

Transforming Escherichia coli BL21(DE3) (Tiangen corporation) from pET-NSBmacpB in the second step by a calcium chloride method, screening and culturing positive clones by kanamycin resistance screening, picking up single clones, carrying out PCR identification by using the P7 and the P8 as primers, and taking the positive clones which are PCR products of about 552bp obtained by PCR identification as gene engineering bacteria and named as pET-NSBmacpB/BL 21. The pET-NSBmacpB/BL 21 strain was selected, inoculated into LB medium containing 100ug/ml kanamycin (a medium obtained by adding kanamycin to LB medium to 100ug/ml kanamycin concentration), and cultured at 37 ℃ to 0D₆₀₀When the value (LB medium containing 100ug/ml kanamycin as blank control) reached 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 resuspending the thallus with 50mM Tris-HCl (pH7.1) to obtain thallus content of 10⁸Suspension of cfu/ml of cellsThe suspension of the cells is subjected to ultrasonication and then centrifuged at 12000 r/min for 10min, and the supernatant (containing total proteins of the cells) is collected and named as NSBmacpB-His crude enzyme solution.

3. Empty vector control bacterium

pET-30b (+) was transformed into E.coli BL21(DE3) in the same manner as in step 1, and the resulting recombinant E.coli was named pET-30b (+)/BL 21. The total bacterial protein was prepared by induction expression of pET-30b (+)/BL21 as an empty vector control by the method described in step 1. The pET-30b (+)/BL21 strain was selected, inoculated into LB medium containing 100ug/ml kanamycin (a medium obtained by adding kanamycin to LB medium to 100ug/ml kanamycin concentration), and cultured at 37 ℃ to 0D₆₀₀When the value (LB medium containing 100ug/ml kanamycin as blank control) reached 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 resuspending the thallus with 50mM Tris-HCl (pH7.1) to obtain thallus content of 10⁸cfu/ml thallus suspension is subjected to ultrasonic disruption, is centrifuged at 12000 r/min for 10min, and supernatant (containing total thallus protein) is collected and named as empty carrier control bacteria crude enzyme liquid.

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) was picked up, inoculated into LB medium containing 100ug/ml kanamycin (a medium obtained by adding kanamycin to LB medium to 100ug/ml kanamycin concentration), and cultured at 37 ℃ to 0D₆₀₀When the value (LB medium containing 100ug/ml kanamycin as blank control) reached 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 resuspending the thallus with 50mM Tris-HCl (pH7.1) to obtain thallus content of 10⁸cfu/ml thallus suspension is subjected to ultrasonic disruption, is centrifuged at 12000 r/min for 10min, and supernatant (containing total thallus protein) is collected and named as blank control bacteria crude enzyme liquid.

Take 30. mu.L of NSBmacpACrude His enzyme solution (from 10)⁸cfu pET-NSBmacpA/BL 21), 30. mu.L NSBmacpB-His crude enzyme solution (from 10)⁸cfu pET-NSBmacpB/BL 21), 30. mu.L of crude enzyme solution of empty vector control bacteria (from 10⁸cfu pET-30b (+)/BL21) and 30. mu.L of the placebo crude enzyme solution (from 10⁸cfu E.coli BL21(DE3)) on the same gel was analyzed by SDS-PAGE (gel concentration of 12%), and the gel was uniform in pore volume and shape and 80. mu.L in pore volume.

The SDS-PAGE results are shown in fig. 1, which indicate that, although there are 27kD bands in each of the crude NSBmacpA-His enzyme solution, the crude NSBmacpB-His enzyme solution, the crude empty vector control bacterium enzyme solution and the crude blank control bacterium enzyme solution, the content of the 27kD polypeptide in the crude NSBmacpA-His enzyme solution and the crude NSBmacpB-His enzyme solution is significantly higher than the content of the 27kD polypeptide in the crude empty vector control bacterium enzyme solution and the crude blank control bacterium enzyme solution, and the content of the 27kD polypeptide in the crude NSBmacpA-His enzyme solution is higher than the content of the 27kD polypeptide in the crude NSBmacpB-His enzyme solution. Shows that both NSBmacpA-His and NSBmacpB-His are expressed in Escherichia coli BL21(DE3), and the expression level of NSBmacpA-His in Escherichia coli BL21(DE3) is higher than that of NSBmacpB-His in Escherichia coli BL21(DE 3).

Fourthly, measuring the phosphohydrolase activity of NSBmacpA-His and NSBmacpB-His

Respectively purifying the NSBmacpA-His crude enzyme solution, the NSBmacpB-His crude enzyme solution, the empty vector control bacteria crude enzyme solution and the blank control bacteria crude enzyme solution obtained in the third step by using a nickel column (a high-affinity Ni-NTA Rasin product purchased 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 NSBmacpA-His shows that 15 amino acids at the N terminal are the 1 st to 15 th amino acids of the sequence 6 in the sequence table, and the sequence determination result of NSBmacpB-His shows that 15 amino acids at the N terminal are the 1 st to 15 th amino acids of the sequence 8 in the sequence table.

Dialyzing the collected eluent by double distilled water, and removing salt ions to respectively obtain pure NSBmacpA-His enzyme solution, pure NSBmacpB-His enzyme solution, pure empty carrier control bacterium enzyme solution and pure blank control bacterium enzyme solution which are used as enzyme solutions 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 phosphohydrolase activity of NSBmacpA-His and NSBmacpB-His was determined by the commonly used sodium p-nitrophenol phosphate (pNPP) method. The adopted reaction system comprises enzyme solution to be detected, sodium p-nitrophenolphosphate (pNPP), 50mmol/L acetic acid-sodium acetate buffer solution and MnCl₂The pH value of the reaction system is 5.0, the concentration of pNPP is 1mmol/L, and MnCl is adopted₂The concentration of (2) was 1 mmol/L. The reaction was carried out at 37 ℃ for 10min, and immediately after the reaction, 0.1ml of 5mmol/L Na0H was added to terminate the reaction and A was measured_405nm. Blank reaction system was used as a blank control. The blank reaction system comprises enzyme solution to be detected, p-nitrophenol sodium phosphate (pNPP), 50mmol/L acetic acid-sodium acetate buffer solution and MnCl which are subjected to heat inactivation and have equal volume₂The pH of the blank reaction system is 5.0, the concentration of pNPP is 1mmol/L, and MnCl₂The concentration of (2) was 1 mmol/L. The enzyme activity unit (U) is defined as: the amount of the phosphohydrolysate pNP (p-nitrophenol) catalytically produced at 37 ℃ and pH5.0 was 1 enzyme activity unit per minute.

The experiment was performed in triplicate. The results showed that the pure empty vector control enzyme solution and the pure blank control enzyme solution had no phosphohydrolase activity, the phosphohydrolase activity of NSBmacpA-His expressed by pET-NSBmacpA/BL 21 was 37.35 ± 1.55U/mg protein, and the phosphohydrolase activity of NSBmacpB-His expressed by pET-NSBmacpB/BL 21 was 12.49 ± 1.26U/mg protein. The phosphohydrolase activity of NSBmacpA-His is 2.99 times that of NSBmacpB-His.

Fifth, Effect of different reaction times and pH on phosphotransferase Activity of NSBmacpA-His and NSBmacpB-His

The phosphotransferase activity of NSBmacpA-His and NSBmacpB-His was demonstrated using inosine conversion rate (ability to convert inosine to inosinic acid). The specific method comprises the following steps:

and taking the pure NSBmacpA-His enzyme liquid and the pure NSBmacpB-His enzyme liquid obtained in the step four 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 ability of NSBmacpA-His and NSBmacpB-His to convert inosine into inosinic acid was determined using 5 reaction systems (pH4.0 reaction system, pH5.0 reaction system, pH6.0 reaction system, pH7.0 reaction system, and pH8.0 reaction system) having different pH values.

The 5 reaction systems with different pH values are composed of enzyme solution to be detected, p-nitrophenol sodium phosphate (pNPP), inosine and buffer solution. The concentrations of pNPP and inosine in the 5 reaction systems with different pH values were all 5mmol/L and 1mmol/L, respectively. pH4.0 the pH of the reaction system was 4.0, and the buffer solution was 0.2mmol/L acetic acid-sodium acetate buffer solution. pH5.0 the pH of the reaction system was 5.0, and the buffer solution was 0.2mmol/L acetic acid-sodium acetate buffer solution. pH6.0 the pH of the reaction system was 6.0, and the buffer solution was 0.2mmol/L sodium dihydrogenphosphate-disodium hydrogenphosphate. pH7.0 the pH of the reaction system was 7.0, and the buffer solution was 0.2mmol/L sodium dihydrogenphosphate-disodium hydrogenphosphate. pH8.0 the reaction system had a pH of 8.0 and the buffer solution was 0.2mmol/L sodium dihydrogen phosphate-disodium hydrogen phosphate.

The reaction systems were reacted at 37 ℃ for 15min, 30min, 45min, 60min, 75min, 90min and 120min, respectively, 0.1ml of 5mmol/L Na0H was added immediately after the reaction to terminate the reaction, and the content of inosinic acid was measured by HPLC analysis, and phosphotransferase activities of NSBmacpA-His and NSBmacpB-His were exhibited according to the inosine conversion rate (inosine conversion rate ═ inosinic acid content/inosine content × 100%). The corresponding blank reaction system was used as a blank control. The blank reaction system of each pH value only replaces the enzyme solution to be detected in the corresponding pH value reaction system with heat-inactivated enzyme solution to be detected with the same volume, and other components are the same as those in the corresponding pH value reaction system. The HPLC conditions were as follows: a chromatographic column: hypersil SAX 5 μm (4.6 mm. times.250 mm), mobile phase: 60mmol/L pH3.0 phosphate-ammonium dihydrogen phosphate buffer, flow rate: 1mL/min, detection wavelength 254nm, column temperature: at 25 ℃.

The experiment was performed in triplicate. The results are shown in fig. 2 and fig. 3, which indicate that the phosphotransferase activity of NSBmacpA-His is at a higher level under acidic conditions (pH4.0-6.0), the inosine conversion rate can reach 31% -38% within 45min, and the inosine conversion rate is highest at 38% at pH5 and reaction time of 30min (fig. 2); the phosphotransferase activity of NSBmacpB-His was lower than that of NSBmacpA-His under acidic conditions (pH4.0-6.0), and the inosine conversion rate of the NSBmacpB-His was up to 20% at pH5 for 30min of the reaction (FIG. 3). The inosine conversion rates of the NSBmacpA-His and the NSBmacpB-His are highest under the conditions of pH value of 5 and reaction time of 30min, the inosine conversion rate of the NSBmacpA-His is higher than that of the NSBmacpB-His, and the inosine conversion rate of the NSBmacpA-His is 1.90 times that of the NSBmacpB-His.

Thus, both NSBmacpA-His and NSBmacpB-His have phosphohydrolase activity and phosphotransferase activity under acidic conditions and are acid phosphatases.

Sixth, Effect of Metal ions on the Activity of NSBmacpA-His and NSBmacpB-His phosphohydrolases

Using 9 different reaction systems (Mn)²⁺Reaction System, Cu²⁺Reaction System, Fe²⁺Reaction System, Zn²⁺Reaction System, Co²⁺Reaction System, Ca²⁺Reaction system, control reaction system and EDTA reaction system) were measured for phosphohydrolase activity of NSBmacpA-His and NSBmacpB-His.

The control reaction system consists of enzyme solution to be detected, p-nitrophenol sodium phosphate (pNPP) and 50mmol/L acetic acid-sodium acetate buffer solution, the pH value of the reaction system is 5.0, and the concentration of the pNPP is 1 mmol/L.

Mn²⁺The reaction system consists of enzyme solution to be detected, sodium p-nitrophenol phosphate (pNPP) and 50mmol/L acetic acid-sodium acetate buffer solution and MnCl₂The pH value of the reaction system is 5.0, the concentration of pNPP is 1mmol/L, and MnCl is adopted₂The concentration of (2) was 1 mmol/L.

Cu²⁺The reaction system consists of enzyme solution to be detected, sodium p-nitrophenol phosphate (pNPP) and 50mmol/L acetic acid-sodium acetate buffer solution and CuCl₂The pH value of the reaction system is 5.0, the concentration of pNPP is 1mmol/L, and CuCl₂The concentration of (2) was 1 mmol/L.

Fe²⁺The reaction system consists of enzyme solution to be detected, sodium p-nitrophenol phosphate (pNPP) and 50mmol/L acetic acid-sodium acetate buffer solution and FeCl₂The pH of the reaction system is 5.0, the concentration of pNPP is 1mmol/L, FeCl₂The concentration of (2) was 1 mmol/L.

Zn²⁺The reaction system consists of enzyme solution to be detected, sodium p-nitrophenol phosphate (pNPP) and 50mmol/L acetic acid-sodium acetate buffer solution and ZnCl₂The pH of the reaction system is 5.0, the concentration of pNPP is 1mmol/L, ZnCl₂The concentration of (2) was 1 mmol/L.

Co²⁺The reaction system consists of enzyme solution to be detected, sodium p-nitrophenol phosphate (pNPP) and 50mmol/L acetic acid-sodium acetate buffer solution and CoCl₂The reaction system has pH of 5.0, pNPP concentration of 1mmol/L, and CoCl₂The concentration of (2) was 1 mmol/L.

Ca²⁺The reaction system consists of enzyme solution to be detected, sodium p-nitrophenol phosphate (pNPP) and 50mmol/L acetic acid-sodium acetate buffer solution and CaCl₂The pH value of the reaction system is 5.0, the concentration of pNPP is 1mmol/L, CaCl₂The concentration of (2) was 1 mmol/L.

The EDTA reaction system consists of enzyme solution to be detected, p-nitrophenol sodium phosphate (pNPP), 50mmol/L acetic acid-sodium acetate buffer solution and EDTA disodium, the pH value of the reaction system is 5.0, the concentration of the pNPP is 1mmol/L, and the concentration of the EDTA is 5 mmol/L.

Each of the above reaction systems was reacted at 37 ℃ for 10min, and immediately after the reaction, 0.1ml of 5mmol/L Na0H was added to terminate the reaction and A was measured_405nm. The corresponding blank reaction system was used as a blank control. The blank reaction system of each reaction system is only to replace the enzyme solution to be detected in the corresponding reaction system with the heat inactivation enzyme solution to be detected with the same volume, and other components are the same as those of the corresponding reaction system. The enzyme activity unit (U) is defined as: the amount of the phosphohydrolysate pNP (p-nitrophenol) catalytically produced at 37 ℃ and pH5.0 was 1 enzyme activity unit per minute.

The results are shown in FIG. 4, except that Ca²⁺+ and EDTA, the addition of divalent metal cations can increase the phosphate water of both NSBmacpA-His and NSBmacpB-HisLyase activity, in which Mn is²⁺、Zn²⁺The best effect of improving the activity of the acid phosphatase is achieved, and Mn is added into NSBmacpA-His in an exogenous way²⁺、Zn²⁺Thereafter, the enzyme activities were 37.35. + -. 1.55U/mg protein and 32.54. + -. 1.36U/mg protein, respectively, while NSBmacpB-His had Mn added exogenously²⁺、Zn²⁺Thereafter, the enzyme activities were 12.49. + -. 1.26U/mg protein and 10.12. + -. 1.17U/mg protein, respectively, and comparison of the two revealed that the phosphohydrolase activity of NSBmacpA-His was 2.99 to 3.22 times that of NSBmacpB-His.

Example 2 cultivation of acid phosphatase phosphate solubilizing engineering bacteria and functional characterization thereof

1. Construction of bacillus megaterium acid phosphatase BmacpA gene shuttle expression vector

In order to obtain a phosphate solubilizing engineering bacterium for efficiently expressing Bacillus megaterium acid phosphatase BmacpA gene, a shuttle expression vector capable of crossing host expression needs to be constructed first. pHT43 (product of MoBiTec, Germany, purchased from Wuhan vast Ling Biotech Co., Ltd.) is a commonly used shuttle expression vector carrying BamHI and XbaI enzyme recognition sites, having ampicillin and chloramphenicol resistance genes, and capable of high-level secretory expression of a target protein by IPTG induction. The gene sequence of BmacpA of bacillus megaterium acid phosphatase is analyzed by DNAMAN software, no BamHI and XbaI enzyme cutting sites are found, and a shuttle expression vector pHT43 can be adopted to construct a recombinant plasmid capable of being expressed in Citrobacter.

Primers were designed based on the coding region sequence of Bacillus megaterium BmacpA gene, BamHI cleavage site (GGATCC) was added to the 5 'primer, and XbaI cleavage site (TCTAGA) was added to the 3' primer. The restriction sites are underlined. The upstream and downstream primers are respectively: p9: 5' -AT-GGATCC-ATGTATGTGA AACGATATCG-3' and P10: 5' -GC-TCTAGA-CTACTTTTGT CGAACACATA-3'). Introducing BamHI and XbaI enzyme recognition sites into the 5 'end and 3' end of the complete coding region of the BmacpA gene respectively by using a PCR amplification method to obtain a BmacpA gene PCR product; the shuttle expression vector pHT43 and BmacpA gene PCR product with enzyme recognition site are simultaneously digested by BamHI and XbaI, and the recovered digestion product is digested by T₄Ligation with ligase, ligation productAfter transformation, positive clones are screened and sequenced, and the sequencing result shows that the DNA molecule shown in the sequence 1 in the sequence table is used for replacing the fragment between the BamHI recognition site and the XbaI recognition site of pHT43 to obtain the recombinant expression vector which is named as pHT-BmacpA. pHT-BmacpA contains BmacpA gene shown in sequence 1 in the sequence table, and pHT-BmacpA expression is protein shown in sequence 2 in the sequence table.

2. Acquisition of acid phosphatase engineering bacteria and phosphohydrolase activity of BmacpA expressed by same

And (3) transferring the recombinant vector pHT-BmacpA into the Citrobacter ACCC02187 by adopting an electrical conversion method to obtain a recombinant bacterium, namely the acid phosphatase engineering bacterium. The acid phosphatase engineering bacteria are inoculated into an LB culture medium containing ampicillin and chloramphenicol and cultured overnight. Inoculating 2% of the strain into LB culture medium containing ampicillin and chloramphenicol, continuing culturing at 35 deg.C to logarithmic phase, adding IPTG to final concentration of 0.5mM, inducing and culturing for 6h, centrifuging at 4000r/min at room temperature for 15min, and collecting supernatant and thallus respectively, wherein the supernatant is extracellular supernatant. Crushing the thallus, centrifuging at the rotation speed of 4000r/min at 4 ℃ for 15min, and collecting supernatant, wherein the supernatant is intracellular supernatant. Then, SDS-PAGE analysis and enzyme activity determination were carried out on the intracellular and extracellular supernatants, respectively. And respectively taking the intracellular supernatant and the extracellular supernatant as enzyme solutions 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 phosphohydrolase activity of the enzyme solution to be tested was determined by the commonly used sodium p-nitrophenol phosphate (pNPP) method. The adopted reaction system comprises enzyme solution to be detected, sodium p-nitrophenolphosphate (pNPP), 50mmol/L acetic acid-sodium acetate buffer solution and MnCl₂The pH value of the reaction system is 5.0, the concentration of pNPP is 1mmol/L, and MnCl is adopted₂The concentration of (2) was 1 mmol/L. The reaction was carried out at 37 ℃ for 10min, and immediately after the reaction, 0.1ml of 5mmol/L Na0H was added to terminate the reaction and A was measured_405nm. Blank reaction system was used as a blank control. The blank reaction system comprises enzyme solution to be detected, p-nitrophenol sodium phosphate (pNPP), 50mmol/L acetic acid-sodium acetate buffer solution and MnCl which are subjected to heat inactivation and have equal volume₂The pH of the blank reaction system is 5.0, the concentration of pNPP is 1mmol/L, and MnCl₂The concentration of (2) was 1 mmol/L. Enzyme activityThe unit (U) is defined as: the amount of the phosphohydrolysate pNP (p-nitrophenol) catalytically produced at 37 ℃ and pH5.0 was 1 enzyme activity unit per minute. The experiment was repeated three times.

SDS-PAGE electrophoresis result (figure 5) shows that the BmacpA gene can be normally expressed in acid phosphatase engineering bacteria, an expression product is a secretory protein, and the molecular weight of the expression product is about 24 kD. The enzyme activity of the phosphohydrolase expressed by the acid phosphatase engineering bacteria is 33.96 +/-1.32U/mg.

3. Effect of reaction time and pH on the phosphotransferase Activity of acid phosphatase engineering bacteria

The phosphotransferase activity of the acid phosphatase engineering bacteria is embodied by the inosine conversion rate (the capability of converting inosine into inosinic acid). The specific method comprises the following steps:

and (3) inoculating the acid phosphatase engineering bacteria in the step (2) into an LB culture medium containing ampicillin and chloramphenicol, and culturing overnight. Transferring the strain with the inoculum size of 2% to LB culture medium containing ampicillin and chloramphenicol, continuing to culture at 35 ℃ until logarithmic phase, adding IPTG until the final concentration is 0.5mM, performing induced culture at 28 ℃ for 6h, centrifuging at 4000r/min at room temperature for 15min, collecting thalli, and determining the capability of converting inosine into inosinic acid by using 5 reaction systems with different pH values (a pH4.0 reaction system, a pH5.0 reaction system, a pH6.0 reaction system, a pH7.0 reaction system and a pH8.0 reaction system).

The 5 reaction systems with different pH values are composed of thalli, p-nitrophenol sodium phosphate (pNPP), inosine and buffer solution. The pNPP concentration, inosine concentration and cell content of the 5 reaction systems with different pH values were all 5mmol/L and 1mmol/L respectively, and the cell content was 10⁸cfu/mL. pH4.0 the pH of the reaction system was 4.0, and the buffer solution was 0.2mmol/L acetic acid-sodium acetate buffer solution. pH5.0 the pH of the reaction system was 5.0, and the buffer solution was 0.2mmol/L acetic acid-sodium acetate buffer solution. pH6.0 the pH of the reaction system was 6.0, and the buffer solution was 0.2mmol/L sodium dihydrogenphosphate-disodium hydrogenphosphate. pH7.0 the pH of the reaction system was 7.0, and the buffer solution was 0.2mmol/L sodium dihydrogenphosphate-disodium hydrogenphosphate. pH8.0 the reaction system had a pH of 8.0 and the buffer solution was 0.2mmol/L sodium dihydrogen phosphate-disodium hydrogen phosphate.

The above reaction systems were reacted at 37 ℃ for 15min, 30min, 45min, 60min, 75min, 90min and 120min, respectively, 0.1ml of 5mmol/L Na0H was added immediately after the reaction to terminate the reaction, and the content of inosinic acid was determined by HPLC analysis, and the phosphotransferase activity of acid phosphatase engineering bacteria was expressed in terms of the inosine conversion rate (inosine conversion rate ═ inosine acid content/inosine content × 100%). The corresponding blank reaction system was used as a blank control. The blank reaction system of each pH value only replaces the enzyme solution to be detected in the corresponding pH value reaction system with heat-inactivated enzyme solution to be detected with the same volume, and other components are the same as those in the corresponding pH value reaction system. The HPLC conditions were as follows: a chromatographic column: hypersil SAX 5 μm (4.6 mm. times.250 mm), mobile phase: 60mmol/L pH3.0 phosphate-ammonium dihydrogen phosphate buffer, flow rate: 1mL/min, detection wavelength 254nm, column temperature: at 25 ℃.

The optimum pH value of the acid phosphatase engineering bacteria for converting inosine into inosinic acid is 5.0, and the inosine conversion rate under an acid condition (pH4.0-6.0) is at a higher level and can reach more than 25 percent. Among them, the conversion rate of inosine by 45 minutes reaction at 37 ℃ and pH5 was 36% (FIG. 6).

4. Phosphate solubilizing effect of acid phosphatase engineering bacteria

Respectively inoculating the acid phosphatase engineering bacteria and the citric acid bacillus (receptor bacteria) in the step 2 into an inositol phosphate liquid culture medium, a phospholipid liquid culture medium and a nucleic acid liquid culture medium, so that the contents of the acid phosphatase engineering bacteria and the citric acid bacillus are 10⁸cfu/mL, culturing at 35 ℃ to logarithmic phase, adding IPTG to a final concentration of 0.5mM, performing induced culture at 28 ℃ for 6h, centrifuging at 4000r/min at room temperature for 15min, collecting supernatant, and directly measuring the content of available phosphorus in the culture solution inoculated with the acid phosphatase engineering bacteria by using a key antimony colorimetric method and a 722 type spectrophotometer at a wavelength of 700 nm.

Wherein, the pH value of the inositol phosphate liquid culture medium is 6.0, 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: 0.3g/L MgSO₄·7H₂O，0.2g/L(NH₄)₂SO₄，0.03g/L CaCl₂，0.9g/LNaCl, 0.5mL of trace element solution/L, and 20g/L of glucose. Wherein, the solvent of the microelement solution is water, and the solutes and the concentrations thereof are as follows: 1.23g/L MnSO₄，0.356g/L ZnSO₄，0.256g/L FeSO₄，0.31g/L CuSO₄·5H₂And

O. Sterilizing

1,4, 5-inositol triphosphate by a bacterial filter, and adding the sterilized culture solution to obtain an inositol phosphate liquid culture medium, wherein the concentration of the 1,4, 5-inositol triphosphate in the inositol phosphate liquid culture medium is 50 mu mol/L.

The pH value of the phospholipid liquid culture medium is 6.0, 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: 0.3g/L MgSO₄·7H₂O，0.2g/L(NH₄)₂SO₄，0.03g/L CaCl₂0.9g/L NaCl, 0.5mL trace element solution/L, 20g/L glucose. Wherein, the solvent of the microelement solution is water, and the solutes and the concentrations thereof are as follows: 1.23g/L MnSO₄，0.356g/L ZnSO₄，0.256g/L FeSO₄，0.31g/L CuSO₄·5H₂And O. Sterilizing L-A-phosphatidylinositol by a bacterial filter, and adding the sterilized culture solution to obtain a phospholipid liquid culture medium, wherein the concentration of the L-A-phosphatidylinositol in the phospholipid liquid culture medium is 50 mu mol/L.

The pH of the nucleic acid liquid culture medium is 6.0, 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: 0.3g/L MgSO₄·7H₂O，0.2g/L(NH₄)₂SO₄，0.03g/L CaCl₂0.9g/L NaCl, 0.5mL trace element solution/L, 20g/L glucose. Wherein, the solvent of the microelement solution is water, and the solutes and the concentrations thereof are as follows: 1.23g/L MnSO₄，0.356g/L ZnSO₄，0.256g/L FeSO₄，0.31g/L CuSO₄·5H₂And O. The salmon sperm DNA is sterilized by a bacterial filter and added into the sterilized culture solution to obtain a nucleic acid liquid culture medium, and the concentration of the salmon sperm DNA in the nucleic acid liquid culture medium is 50 mu mol/L. As a result, it was revealed that the culture was carried out in a nucleic acid liquid medium (containing salmon sperm DNA) or a phospholipid liquid medium (containing L-A-phosphatidyl group) as compared with the case of Citrobacter as a recipient bacteriumInositol) and inositol phosphate liquid medium (containing 1,4, 5-triphosphate inositol), the content of available phosphorus was increased by 14.59. mu. mol/L, 16.63. mu. mol/L and 18.55. mu. mol/L, respectively (FIG. 7).

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

<120> method for constructing recombinant microorganism having phosphorus-solubilizing activity and nucleic acid molecule used therefor

<130> GNCFH181963

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<170> PatentIn version 3.5

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atgtttaata caccttgggt gaaacagctt gactttaatg tacttcatac cattgaagga 60

tggaggacgg acacgttaac acctatcatc atatttataa caaccgtagg atcttggtat 120

gtcactgcgc caatatggtt tgcaattata gtatttcttc tttataaacg aaaaggattg 180

ctcgctttat atatcacgct cgttttttgg ggagttcgcg ctttaaattg gggattgaag 240

gagatttttg caagaccaag acctgattgg agtcaagtcg ttcccgcctc tcactatagt 300

tttccgagtg gacatgccat gaactcaatg gcgttttaca gcggaatact tttgttaata 360

tggatgtata caagaagcag ggctgttaaa acggcagctg catgcgtaat agctattgtt 420

attttattaa ttggatttag ccgtttgtat ttaggggtac actttttaac ggatatacta 480

gcaggatatt gtttaggact tgtttggtcc ttaggagtct atctcctttc taagcgctta 540

tataaccaaa aaaagcttgc ggccgcactc gagcaccacc accaccacca ctga 594

<210> 8

<211> 197

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Met Phe Asn Thr Pro Trp Val Lys Gln Leu Asp Phe Asn Val Leu His

1 5 10 15

Thr Ile Glu Gly Trp Arg Thr Asp Thr Leu Thr Pro Ile Ile Ile Phe

20 25 30

Ile Thr Thr Val Gly Ser Trp Tyr Val Thr Ala Pro Ile Trp Phe Ala

35 40 45

Ile Ile Val Phe Leu Leu Tyr Lys Arg Lys Gly Leu Leu Ala Leu Tyr

50 55 60

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

65 70 75 80

Glu Ile Phe Ala Arg Pro Arg Pro Asp Trp Ser Gln Val Val Pro Ala

85 90 95

Ser His Tyr Ser Phe Pro Ser Gly His Ala Met Asn Ser Met Ala Phe

100 105 110

Tyr Ser Gly Ile Leu Leu Leu Ile Trp Met Tyr Thr Arg Ser Arg Ala

115 120 125

Val Lys Thr Ala Ala Ala Cys Val Ile Ala Ile Val Ile Leu Leu Ile

130 135 140

Gly Phe Ser Arg Leu Tyr Leu Gly Val His Phe Leu Thr Asp Ile Leu

145 150 155 160

Ala Gly Tyr Cys Leu Gly Leu Val Trp Ser Leu Gly Val Tyr Leu Leu

165 170 175

Ser Lys Arg Leu Tyr Asn Gln Lys Lys Leu Ala Ala Ala Leu Glu His

180 185 190

His His His His His

195

Claims

1. The application of nucleic acid molecules in constructing microorganisms with phosphorus-solubilizing activity; the nucleic acid molecule encodes a protein of a) or b) or c):

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

2. A method for constructing a recombinant microorganism having a phosphorus solubilizing activity, which comprises introducing a gene encoding the protein of a) or b) or c) of claim 1 into a recipient microorganism to obtain a recombinant microorganism having a phosphorus solubilizing activity higher than that of the recipient microorganism.

3. The method of claim 2, wherein: the coding gene is a DNA molecule shown in the following 1) or 2) or 3):

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

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

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

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

6. The method of claim 5, wherein: the gram-negative bacterium is an escherichia bacterium or a citrobacter bacterium.

7. The recombinant microorganism having a phosphorus solubilizing activity constructed by the method of any one of claims 2 to 6.