Gene, recombinant expression vector, strain of 3-hydroxybenzoic acid 2-hydroxylase and method for hydroxylating hydroxybenzoic acid

Abstract

The invention discloses a gene of 3-hydroxybenzoic acid 2-hydroxylase, a recombinant expression vector, a strain and a hydroxylation method of hydroxybenzoic acid, and relates to the technical field of genetic engineering. The gene of the 3-hydroxybenzoic acid 2-hydroxylase, the nucleotide sequence of the gene of the 3-hydroxybenzoic acid 2-hydroxylase is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. The gene of the 3-hydroxybenzoic acid 2-hydroxylase provided by the invention can encode the 3-hydroxybenzoic acid 2-hydroxylase, and the obtained 3-hydroxybenzoic acid 2-hydroxylase can effectively catalyze the 2'-position hydroxylation of 3-hydroxybenzoic acid to generate 2,3-dihydroxybenzoic acid, thereby realizing the selective hydroxylation of aromatic compounds.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a 3-hydroxybenzoate 2-hydroxylase gene, a recombinant expression vector, a strain and a hydroxylation method of hydroxybenzoic acid. Background Art

[0002] The selective hydroxylation of aromatic compounds is one of the most challenging reactions in synthetic chemistry. Hydroxylated aromatic compounds, in particular, have attracted widespread attention as drug precursors. Hydroxybenzoic acid is a common compound metabolized in animals, plants, and microorganisms. It is also used as an intermediate in the synthesis of resins and pharmaceuticals, as well as a corrosion inhibitor. Hydroxylation of hydroxybenzoic acid includes 6'-hydroxylation catalyzed by 3-hydroxybenzoate 6-hydroxylase, 4'-hydroxylation catalyzed by 3-hydroxybenzoate 4-hydroxylase, and 2'-hydroxylation catalyzed by 3-hydroxybenzoate 2-hydroxylase. However, only enzymatic activity has been detected for hydroxybenzoate 2-hydroxylase, and the coding gene and protein sequence are lacking, which has hindered its application. Summary of the Invention

[0003] The main purpose of the present invention is to provide a 3-hydroxybenzoate 2-hydroxylase gene, a vector, a recombinant expression strain and a hydroxylation method for hydroxybenzoic acid, aiming to efficiently catalyze the hydroxylation of hydroxybenzoic acid.

[0004] To achieve the above object, the present invention provides a 3-hydroxybenzoate 2-hydroxylase gene, the nucleotide sequence of the 3-hydroxybenzoate 2-hydroxylase gene is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

[0005] The present invention also provides a recombinant expression vector comprising the above-mentioned 3-hydroxybenzoate 2-hydroxylase gene.

[0006] The present invention also provides a recombinant expression strain comprising the gene of 3-hydroxybenzoate 2-hydroxylase as described above.

[0007] Preferably, the host cell of the recombinant expression strain is Escherichia coli.

[0008] The present invention also provides a method for cloning the gene of 3-hydroxybenzoate 2-hydroxylase as described above, comprising the following steps:

[0009] Screening of 3-hydroxybenzoic acid and 2,3-dihydroxybenzoic acid utilizing strains from environmental soil samples;

[0010] Performing genome sequencing on the strain to obtain genome information;

[0011] Genome annotation and the gene for 3-hydroxybenzoate 2-hydroxylase were obtained.

[0012] The present invention also provides a method for expressing the gene of 3-hydroxybenzoate 2-hydroxylase as described above, comprising the following steps:

[0013] The gene of 3-hydroxybenzoate 2-hydroxylase is cloned into an expression vector to obtain a recombinant expression vector;

[0014] Transforming the recombinant expression vector into a host cell to obtain a recombinant expression strain;

[0015] The recombinant expression strain was cultured, and 3-hydroxybenzoate 2-hydroxylase was obtained from the culture.

[0016] The present invention also provides a method for hydroxylating hydroxybenzoic acid, comprising the following steps: using the 3-hydroxybenzoic acid 2-hydroxylase expressed by the 3-hydroxybenzoic acid 2-hydroxylase gene to catalyze the hydroxylation of hydroxybenzoic acid.

[0017] The 3-hydroxybenzoate 2-hydroxylase gene provided by the present invention can encode 3-hydroxybenzoate 2-hydroxylase. The obtained 3-hydroxybenzoate 2-hydroxylase can effectively catalyze the 2'-hydroxylation of 3-hydroxybenzoic acid to produce 2,3-dihydroxybenzoic acid, thereby achieving selective hydroxylation of aromatic compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 This is a sequence verification diagram of the pET28a-mhboA recombinant expression vector constructed in Example 2 of the present invention using primer T7er;

[0020] Figure 2 This is a sequence verification diagram of the pET28a-mhboA recombinant expression vector constructed in Example 2 of the present invention using primer T7;

[0021] Figure 3 This is an LC-MS identification analysis diagram of the catalytic product of 3-hydroxybenzoate 2-hydroxylase in Example 5 of the present invention. DETAILED DESCRIPTION

[0022] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0023] It should be noted that, in the embodiments, those without specifying specific conditions, are carried out according to conventional conditions or the conditions recommended by the manufacturer. Those for reagents or instruments used that do not specify the manufacturer are conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes. Taking "A and / or B" as an example, it includes schemes A, B, or A and B that meet the same time. In addition, the technical solutions between the various embodiments can be combined with each other, but must be based on those of ordinary skill in the art. When the combination of the technical solutions is contradictory or cannot be realized, it should be considered that the combination of such technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work belong to the scope of protection of the present invention.

[0024] The selective hydroxylation of aromatic compounds is one of the most challenging reactions in synthetic chemistry. Hydroxylated aromatic compounds, in particular, have attracted widespread attention as drug precursors. Hydroxybenzoic acid is a common compound metabolized in animals, plants, and microorganisms. It is also used as an intermediate in the synthesis of resins and pharmaceuticals, as well as a corrosion inhibitor. Hydroxylation of hydroxybenzoic acid includes 6'-hydroxylation catalyzed by 3-hydroxybenzoate 6-hydroxylase, 4'-hydroxylation catalyzed by 3-hydroxybenzoate 4-hydroxylase, and 2'-hydroxylation catalyzed by 3-hydroxybenzoate 2-hydroxylase. However, only enzymatic activity has been detected for hydroxybenzoate 2-hydroxylase, and the coding gene and protein sequence are lacking, which has hindered its application.

[0025] In view of this, the present invention proposes a gene for 3-hydroxybenzoate 2-hydroxylase, which is intended to efficiently catalyze the hydroxylation of hydroxybenzoate.

[0026] The 3-hydroxybenzoate 2-hydroxylase gene proposed by the present invention has a nucleotide sequence as shown in SEQ ID NO: 1 and an amino acid sequence as shown in SEQ ID NO: 2.

[0027] The sequence of SEQ ID NO: 1 is as follows:

[0028] ATGAGCAAACCAAGCGTCACGGCCAGCCGTGCAGCGTGCCTGTGTGTC

[0029] ACGCTAGTTTTCACCTCGATGACTTACGCCACTGAAGGGGGTGGTAGTT

[0030] CTTATCCTCTCGGTGTTAACACGGTGGGCGCTGGCAAGATGCCACCGCC

[0031] CGGATTTACCGAGTTCTTCTATCTTTCCGACTATCGGGCGAGCCGAACCC

[0032] TCGACGGCGACGGCGATAGAAAAGCCGGCATTCATGACTTCGACCTGAA

[0033] TATCCAGGCACTTTCGATTCGTGTCGATTATGTCTACCAGGACGTTTCATT

[0034] TTTCGGTGCCAAACTTGCCAGTCGCGTCGCCTTGCCACTGGTCAAAGGC

[0035] GATATCAGTTTCAATGTCGATACACCGGCAGGCCGCGTCAGGCGCAGTG

[0036] ACCATCAGGAGGGCGTCGGCGACCTTACCGTCGTGCCATTCGTGCTGGG

[0037] CTGGAGCTCCCCTCGTTACCATCAGCTGTTCGGGCTGGATGTGTTTGTGC

[0038] CGGTGGGTTCCTACGACAAAGACCGCTTGTTCAACCCTGGCAGGAACA

[0039] CCTGGGCCTATGGGCCATGGTATTCATTTACGGCCTATCCGCTGGAAAAC

[0040] CTCGAAGTCAGCGCAAAGCTGATTTACATGATCAACGGGGAGAACAAA

[0041] GATACCGATTATCGCTCCGGTCATGAGTTCAATGCTGACTACAATATTGGC

[0042] TACAACATTACCCGCGAATGGCAGCTTGGGCTAAACGGCTATCTCTACAA

[0043] ACAGGTCAGCGATGACGAAAAAGACGGGCATACCTACCTGGATGGCAAT

[0044] CGGGGTCAAGTGGCCGCAATCGGTCCTGCGTTGAAGTACCAAACCCCC

[0045] GAGTTCGGTTTCGTCATGAAGTGGCAACATGAAACGCAGGTGGAGAATC

[0046] GGGCGGCGGGTGACCGTATTCTGGTTGCAAGCCGTTTTACCGCTTCTGA

[0047] The sequence of SEQ ID NO: 2 is as follows:

[0048] MSKPSVTASRAACLCVTLVFTSMTYATEGGGSSYPLGVNTVGAGKMPPPGFTEFFYLSDYRASRTLDGDGDRKAGIHDFDLNIQALSIRVDYVYQDVSFFGAKLASRVALPLVKGDISFNVDTPAGRVRRSDHQEGVGDLTVVPFVLGWSSPRYH QLFGLDVFVPVGSYDKDRLFNPGRNTWAYGPWYSFTAYPLENLEVSAKLIYMINGENKDTDYRSGHEFNADYNIGYNITREWQLGLNGYLYKQVSDDEKDGHTYLDGNRGQVAAIGPALKYQTPEFGFVMKWQHETQVENRAAGDRIWLQAVYRF

[0049] The 3-hydroxybenzoate 2-hydroxylase gene provided by the present invention can encode 3-hydroxybenzoate 2-hydroxylase. The obtained 3-hydroxybenzoate 2-hydroxylase can effectively catalyze the 2'-hydroxylation of 3-hydroxybenzoic acid to produce 2,3-dihydroxybenzoic acid, thereby achieving selective hydroxylation of aromatic compounds.

[0050] The present invention also provides a 3-hydroxybenzoic acid and 2,3-dihydroxybenzoic acid utilizing strain, comprising the above-mentioned 3-hydroxybenzoic acid 2-hydroxylase gene.

[0051] After screening 3-hydroxybenzoic acid and 2,3-dihydroxybenzoic acid utilizing strains from environmental soil samples, their genomic information was obtained by sequencing, and the gene of 3-hydroxybenzoate 2-hydroxylase was annotated.

[0052] The present invention also provides a recombinant expression vector comprising the above-mentioned 3-hydroxybenzoate 2-hydroxylase gene.

[0053] After obtaining the gene of 3-hydroxybenzoate 2-hydroxylase, the target gene needs to be introduced into the host cell through a vector so that the target gene is replicated as the host cell reproduces, thereby obtaining a large amount of the target gene.

[0054] Recombinant expression vectors are vectors that add expression elements (such as promoters, RBS, terminators, etc.) to the basic skeleton of a cloning vector to enable the expression of the target gene. The promoter type can be a strong expression promoter, a tissue-specific promoter, or an inducible promoter. In practice, the corresponding promoter can be selected to drive expression according to the actual situation.

[0055] The present invention does not limit the type of expression vector. Preferably, in the embodiment of the present invention, the expression vector is pET28a(+), which has a strong promoter and high expression level.

[0056] The present invention also provides a recombinant expression strain comprising the gene of 3-hydroxybenzoate 2-hydroxylase as described above.

[0057] The above-mentioned recombinant expression vector is introduced into a host cell to obtain a recombinant expression strain, and the target gene can be replicated as the host cell reproduces. Generally speaking, the host cell can be Escherichia coli, yeast, or other types of cells such as animal cells.

[0058] Preferably, the host cell of the recombinant expression strain is Escherichia coli.

[0059] Escherichia coli (E. coli) is a representative bacterium of the genus Escherichia. It is generally non-pathogenic and a common resident of the human and animal intestines. As a host for exogenous gene expression, E. coli has attracted considerable attention from genetic engineering experts due to its clear genetic background, simple technical operations, simple culture conditions, and economical large-scale fermentation. Currently, E. coli is the most widely used and successful expression system, often the preferred system for high-efficiency expression.

[0060] Specifically, the E. coli is E. coli BL21(DE3), which is used to efficiently express genes cloned into expression vectors (such as the pET series) containing a bacteriophage T7 promoter. The T7 phage RNA polymerase is located in the DE3 region of lambda phage, which is integrated into the chromosome of BL21. This bacteriophage is suitable for expressing non-toxic proteins.

[0061] The present invention also provides a method for expressing the gene of 3-hydroxybenzoate 2-hydroxylase as described above, comprising the following steps:

[0062] Step S10, cloning the 3-hydroxybenzoate 2-hydroxylase gene into an expression vector to obtain a recombinant expression vector;

[0063] Step S20, transferring the recombinant expression vector into a host cell to obtain a recombinant expression strain;

[0064] Step S30: Cultivate the recombinant expression strain and obtain 3-hydroxybenzoate 2-hydroxylase from the culture.

[0065] The present invention provides a method for expressing a 3-hydroxybenzoate 2-hydroxylase gene. The method comprises cloning the 3-hydroxybenzoate 2-hydroxylase gene into an expression vector pET28a(+), transforming the gene into a host cell, Escherichia coli BL21(DE3), culturing the recombinant expression strain, and obtaining 3-hydroxybenzoate 2-hydroxylase from the culture. The obtained 3-hydroxybenzoate 2-hydroxylase can effectively catalyze the 2'-hydroxylation of 3-hydroxybenzoic acid to produce 2,3-dihydroxybenzoic acid, thereby achieving selective hydroxylation of aromatic compounds.

[0066] The present invention also provides a method for hydroxylating hydroxybenzoic acid, comprising the following steps: using the 3-hydroxybenzoic acid 2-hydroxylase expressed by the 3-hydroxybenzoic acid 2-hydroxylase gene to catalyze the hydroxylation of hydroxybenzoic acid.

[0067] The method for hydroxylating hydroxybenzoic acid proposed by the present invention uses the 3-hydroxybenzoate 2-hydroxylase expressed by the gene of the 3-hydroxybenzoate 2-hydroxylase described above to catalyze the hydroxylation of hydroxybenzoic acid. The method for hydroxylating hydroxybenzoic acid proposed by the present invention uses the 3-hydroxybenzoate 2-hydroxylase expressed by the gene of the 3-hydroxybenzoate 2-hydroxylase described above to catalyze the hydroxylation of hydroxybenzoic acid.

[0068] The technical solutions of the present invention are further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0069] Example 1

[0070] The gene encoding 3-hydroxybenzoate 2-hydroxylase is obtained by the following method:

[0071] (1) Environmental soil samples were inoculated into liquid inorganic salt culture medium (components: Na2HPO4·12H2O 10.2-14.3 g / L, KH2PO4 2-3 g / L, FeSO4·7H2O 0.2-0.3 mg / L, MnSO4·H2O 0.05-0.07 mg / L, MgSO4·7H2O0.015-0.02 mg / L, CaCl2 0.25-0.3 mg / L, CuSO4 0.0125-0.2 mg / L, ZnSO4 0.0125-0.2 mg / L and H3BO3 0.0125-0.2 mg / L, (NH4)2SO4 2-3 mM, the pH value of the liquid inorganic salt culture medium is 7.0-7.5), 3-hydroxybenzoic acid or 2,3-dihydroxybenzoic acid is added, and after shaking culture for 3-5 days, the culture is taken out and inoculated into a new liquid inorganic salt culture medium, and 3-hydroxybenzoic acid or 2,3-dihydroxybenzoic acid is added for subculture;

[0072] (2) Dilute the culture after 3-4 subcultures to a diluted bacterial solution, spread the diluted bacterial solution on an inorganic salt solid culture medium plate, add 3-hydroxybenzoic acid or 2,3-dihydroxybenzoic acid, and culture in an incubator at 28°C to 30°C for 2-3 days;

[0073] (3) A larger single colony on the inorganic salt solid culture medium was picked, and after separation and purification, a screening strain was obtained. The 16S rDNA sequencing results showed that the strain belonged to the genus Pseudomonas and was named CHJ03 (Rhodococcus sp. CHJ03).

[0074] (4) The above strain was sequenced using the PacBio sequencing platform to obtain genome information; the genome was annotated using Glimmer (v3.02) software to obtain the gene of 3-hydroxybenzoate 2-hydroxylase.

[0075] Example 2

[0076] The recombinant expression vector pET28a(+) and the recombinant expression strain Escherichia coli BL21(DE3) were constructed as follows:

[0077] (1) The genome of Pseudomonas sp. CHJ03 was extracted and used as a template for PCR amplification of the 3-hydroxybenzoate 2-hydroxylase gene mhboA. The primers used were: P1: CCGCGCGGCAGCCATATGAGCAAACCAAGCGTCACGG, P2: TCGAGTGCGGCCGCACAGAAGCGGTAAACGGCTTGC. The PCR system was as follows: 30-50 ng template, 2 μl of primers (each 10 μM), 5 μl of 10× EasyPfu Buffer, 1 μl of 10 mM dNTPs, 1 μl of EasyPfu DNA Polymerase (2.5 units), and ddH2O to make up to 50 μl. EasyPfu DNA Polymerase was purchased from Beijing Quanshijin Biotechnology Co., Ltd. PCR reaction conditions were: 94°C pre-denaturation for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 2 min; and then extension at 72°C for 5 min.

[0078] (2) The gene obtained by PCR was ligated to the expression vector pET28a(+) (Novagen, USA) digested with NdeI and HindIII using the ClonExpress II One Step Cloning Kit (Nanjing Novagen Biotechnology Co., Ltd.) to obtain the pET28a-mhboA vector; the enzyme digestion system and ligation system were carried out according to the product instructions.

[0079] (3) The pET28a-mhboA recombinant expression vector was transformed into Escherichia coli BL21 (DE3) (Novagen, USA) by chemical transformation, and then positive transformants were screened on LB medium plates (containing 50 μg / mL of kanamycin). The correct positive transformants were verified by sequencing, and the recombinant expression strain of the 3-hydroxybenzoate 2-hydroxylase gene was obtained.

[0080] The constructed pET28a-mhboA recombinant expression vector was sequenced and verified using universal primers T7er and T7. Figure 1 (T7er) and Figure 2 (T7), indicating that the mhboA gene has been successfully ligated into the expression vector pET28a(+).

[0081] Example 3

[0082] The preparation method of crude 3-hydroxybenzoate 2-hydroxylase enzyme solution is as follows:

[0083] (1) The recombinant expression strain of the 3-hydroxybenzoate 2-hydroxylase gene mhboA in Example 2 (10 mL seed solution) was inoculated into 1 L LB liquid medium (containing 50 μg / mL of kanamycin) for fermentation and culture at 37°C and 200 rpm for about 2 h. The OD 600 When the concentration reaches 0.4-0.6, add 0.1 mM IPTG and culture at 37°C and 200 rpm for 12-16 h to induce expression.

[0084] (2) The induced expression bacterial solution was centrifuged at 8000 rpm for 5 min at 4°C, the supernatant was discarded, and the bacterial cells were suspended in 100 mL of 40 mM HEPES-KOH buffer (pH 8.0), disrupted by ultrasonication, and then centrifuged at 12000 rpm for 50 min at 4°C. The supernatant was collected to obtain the crude 3-hydroxybenzoate 2-hydroxylase enzyme solution.

[0085] Example 4

[0086] The activity of 3-hydroxybenzoate 2-hydroxylase was determined by UV spectrophotometry. The method is as follows:

[0087] The reaction was initiated by adding the substrate. Consumption of the substrate 3-hydroxybenzoate was determined by a decrease in the cofactor NADH or NADPH at 340 nm. The reaction system consisted of 0.2 mM 3-hydroxybenzoate, 0.3 mM NADH or NADPH, 0.04 mM FAD, and 108 μg of the crude enzyme solution described above. The reaction was carried out in 40 mM HEPES–KOH buffer (pH 8.0) at room temperature (same for 20-25°C). Specific activity was determined based on the characteristic absorption peak of the coenzyme NADPH at 340 nm (λmax = 340 nm, molar extinction coefficient = 6,220 M). -1 cm -1 ) is measured by the reduction of the substrate. The control system contains all of these compounds except the substrate 3-hydroxybenzoic acid. The UV spectrum at 230-400 nm is monitored every minute using a Lambda 25 UV / Vis spectrometer (PerkinElmer, USA). A unit of enzyme activity is defined as the amount of enzyme required to reduce 1 μmol of substrate or increase product per minute at room temperature (same below 20-25°C). The specific activity of the enzyme is expressed as the number of enzyme activity units per gram of protease. The specific activity of the enzyme for 3-hydroxybenzoic acid is 172.96 U / mg.

[0088] Example 5

[0089] Identification of the catalytic product of 3-hydroxybenzoate 2-hydroxylase. The method is as follows:

[0090] A 10 mL reaction system was prepared according to Example 4, and an equal volume of methanol was added to terminate the reaction. The mixture was then centrifuged at 12,000 rpm for 2 min, and the supernatant was collected for liquid chromatography-mass spectrometry (LC-MS) analysis.

[0091] LC-MS analysis: LC-MS was performed using an Agilent Triple Quad LC / MS 6460 liquid chromatography-mass spectrometer (Agilent, USA) and an Accucore column. TM A C18 LC column (2.6 μm × 4.6 mm × 100 mm) (Agilent) was used at 30°C, with an injection volume of 10 μL and a flow rate of 0.5 mL / min. The mobile phase consisted of solvent A (water + 0.1% acetic acid): solvent B (methanol + 0.1% acetic acid) = 55%:45%. A triple quadrupole mass spectrometer was used in ESI negative ion mode; the impact voltage was 135.0 V; and the mass scan range was 10–200.

[0092] LC-MS results showed that Figure 3 As shown, in the 3.080 min peak of the total ion chromatogram (TIC), substances with molecular weights of 137.0 and 153.0 were detected. This is consistent with the molecular weights of 3-hydroxybenzoic acid (molecular weight 138) and 2,3-dihydroxybenzoic acid (molecular weight 154) in negative ion mode. Fragment ions (molecular weight 108.9) of 3-hydroxybenzoic acid (molecular weight 138) were also found, such as the molecular weight 109 corresponding to the removal of a carboxyl group in negative ion mode. Therefore, hydroxybenzoate hydroxylase catalyzes the conversion of 3-hydroxybenzoic acid to 2,3-dihydroxybenzoic acid, and it is a 3-hydroxybenzoate 2-hydroxylase.

[0093] The above are only preferred embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A gene for 3-hydroxybenzoate 2-hydroxylase, It is characterized in that The nucleotide sequence of the 3-hydroxybenzoate 2-hydroxylase gene is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO:

2.

2. A recombinant expression vector, It is characterized in that The invention comprises the gene of 3-hydroxybenzoate 2-hydroxylase as claimed in claim 1.

3. A recombinant expression strain, It is characterized in that The invention comprises the gene of 3-hydroxybenzoate 2-hydroxylase as claimed in claim 1.

4. The recombinant expression strain according to claim 3, It is characterized in that The host cell of the recombinant expression strain is Escherichia coli.

5. A method for expressing the gene of 3-hydroxybenzoate 2-hydroxylase as claimed in claim 1, It is characterized in that The following steps are involved: Cloning the gene of 3-hydroxybenzoate 2-hydroxylase into an expression vector to obtain a recombinant expression vector; Transforming the recombinant expression vector into a host cell to obtain a recombinant expression strain; The recombinant expression strain was cultured and 3-hydroxybenzoate 2-hydroxylase was obtained from the culture.

6. A method for hydroxylating hydroxybenzoic acid, It is characterized in that The following steps are involved: The 3-hydroxybenzoate 2-hydroxylase expressed by the 3-hydroxybenzoate 2-hydroxylase gene as claimed in claim 1 is used to catalyze the hydroxylation of hydroxybenzoate.

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