Phenazine halogenase PezW and application thereof

Abstract

The invention discloses the phenazine halogenase PezW for the first time; the halogenase PezW is derived from streptomyces tubercidicus NBRC 13090, and belongs to a flavin dependent type halogenase, and the flavin dependent type halogenase PezW is a flavin dependent type halogenase. The invention also constructs an expression vector containing a nucleotide sequence for coding the phenazine halogenase PezW, a cloning expression method of the phenazine halogenase PezW, and an application of the phenazine halogenase PezW in preparation of halogenated modified compounds. Wherein the compound is hydroxylated phenazine. The halogenation modification is as follows: halogen atoms of a halogen donor are transferred and combined to a phenazine compound; and the halogen donor is NaCl, NaBr or NaI. According to the halogenase PezW, hydroxylated phenazine can be used as a substrate, various halogenated hydroxyphenazine can be obtained, the technical blank of preparing hydroxylated phenazine by an enzyme method is filled, and a remarkable technical effect is achieved. The method not only has milestone significance for the technical development of enzymatic synthesis of HHPs, but also provides a new path for the drug development of phenazine compounds, and has important application value and social significance.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and biosynthesis, and relates to a halogenase, in particular to an enzyme used for halogenation modification of hydroxyphenazine compounds. Background Art

[0002] Halogen atoms possess strong electronegativity, and their presence can enhance the biological activity of compounds and alter their physicochemical properties. Consequently, the widespread application of halogen elements in pharmaceuticals, pesticides, and novel materials has garnered widespread attention from both the scientific and industrial communities. Due to environmental concerns surrounding the chemical synthesis of halogenated organic compounds, halogenases within the biosynthetic pathway of natural halides have garnered increasing attention. Halogenases are enzymes that catalyze the formation of stable carbon-halogen bonds from halogen ions. Currently discovered halogenases are primarily classified into four types based on their mechanism of action and coenzymes (cofactors): haloperoxidases, α-ketoglutarate-dependent halogenases, flavin-dependent halogenases, and S-adenosylmethionine-dependent fluorinases. Most halogenases utilize chlorine, bromine, or iodine as their halogen donor, with chlorine being the most common. Only S-adenosylmethionine-dependent halogenases are fluorinases, utilizing fluorine exclusively as their halogen donor.

[0003] Phenazines are nitrogen-containing aromatic heterocyclic antibiotics derived from microorganisms. Over 100 natural phenazine products have been discovered, exhibiting broad-spectrum antibacterial and antimicrobial activities. The diversity of natural phenazine compounds is primarily achieved through a variety of post-modification enzymes. Gebhardt et al. isolated the isoprenyl-modified phenazine derivatives Endophenazines AD from the metabolites of Streptomycetes anulatus, of which Endophenazine A and Endophenazine D exhibited excellent antibacterial activity. Wang et al. also isolated Nexphenazine A, a phenazine-polyketide hybrid compound, from Streptomyces sp. KIB-H483. Furthermore, halogenated phenazines, and more complex phenazine compounds composed of substituents such as oxymethyl, ester, amino, and sugar groups, have been found in metabolites of Streptomyces and Pseudomonas, many of which are effective antibacterial and antitumor substances.

[0004] Among them, halogenated phenazines (HPs) exhibit significant antibacterial activity. To date, researchers have isolated and identified eight halogenated phenazines from natural products, including seven chlorinated and brominated products of terpenized phenazines and one brominated product of hydroxylated phenazines (2-bromo-1-hydroxyphenazine). Among them, 2-bromo-1-hydroxyphenazine exhibited significant anti-biofilm activity against Staphylococcus aureus and Staphylococcus epidermidis, with an MIC value of 6.25 μM. In addition, according to literature reports, halogenated derivatives of various hydroxylated phenazines (HHPs) have exhibited significant anti-biofilm activity.

[0005] Currently, halogenated derivatives of hydroxylated phenazines are primarily obtained through direct fermentation and chemical total synthesis, with no studies demonstrating the enzymatic preparation of HHPs. Therefore, the halogenation of HHPs enzymatically could provide a new pathway for the development of phenazines as pharmaceuticals, with significant application value and social significance. Summary of the Invention

[0006] To address the existing technical gap in the enzymatic preparation of halogenated derivatives of hydroxyphenazine compounds, this invention discloses for the first time a phenazine halogenase, PezW. Derived from Streptomyces tubercidicus NBRC 13090, PezW is a flavin-dependent halogenase. This halogenase can use hydroxylated phenazines as substrates to produce a variety of halogenated hydroxyphenazines, laying the foundation for the application of phenazine compounds in drug development and possessing significant practical value.

[0007] The technical solution of the present invention:

[0008] A phenazine halogenase PezW, wherein the amino acid sequence of the phenazine halogenase PezW is selected from the following (1), (2) or (3):

[0009] (1) the amino acid sequence shown in SEQ ID NO: 1;

[0010] (2) an amino acid sequence as shown in SEQ ID NO: 1, wherein one or more amino acids are substituted, deleted, or added and the amino acid sequence is capable of catalyzing the transfer of a halogen atom to a hydroxylated phenazine;

[0011] (3) An amino acid sequence having a homology of ≥90% with the amino acid sequence shown in SEQ ID NO: 1, and the expressed protein is capable of catalyzing the transfer of a halogen atom to a hydroxylated phenazine.

[0012] The present invention discloses phenazine halogenase PezW for the first time, realizes the enzymatic halogenation of hydroxylated phenazines, fills the technical gap in the enzymatic preparation of hydroxylated phenazines, and produces significant technical effects.

[0013] The DNA molecule encoding the phenazine halogenase PezW has a nucleotide sequence selected from the following (1), (2), (3) or (4):

[0014] (1) the nucleotide sequence shown in SEQ ID NO: 2;

[0015] (2) a nucleotide sequence that is different from the nucleotide sequence shown in SEQ ID NO: 2 but encodes the amino acid sequence shown in SEQ ID NO: 1;

[0016] (3) a nucleotide sequence that has a homology of ≥85% with the nucleotide sequence shown in SEQ ID NO: 2, and the protein expressed by the nucleotide sequence can catalyze the transfer of a halogen atom to a hydroxylated phenazine;

[0017] (4) A nucleotide sequence complementary to the nucleotide sequence described in any one of (1), (2) or (3).

[0018] An expression vector containing the nucleotide sequence encoding the phenazine halogenase PezW is suitable for expression in Escherichia coli.

[0019] The invention relates to an isolated recombinant vector, which is an expression vector containing a nucleotide sequence encoding the phenazine halogenase PezW.

[0020] A host cell comprising the isolated recombinant vector as described above; the host cell does not include animal or plant species.

[0021] The cloning and expression method of the phenazine halogenase PezW as described above comprises the following steps: cloning the nucleotide sequence encoding the phenazine halogenase PezW into an expression vector to construct an expression vector; then transferring the expression vector into an expression system for expression; and finally purifying to obtain the phenazine halogenase PezW.

[0022] The aforementioned phenazine halogenase PezW is used to prepare halogenated compounds. The compound is a hydroxylated phenazine, specifically 1-hydroxyphenazine, 2-hydroxyphenazine, or 1,6-dihydroxyphenazine. The halogenation modification involves transferring and binding a halogen atom from a halogen donor to the phenazine compound; the halogen donor is NaCl, NaBr, or NaI.

[0023] Preferably, the reaction system of the enzyme-catalyzed reaction is: 50 mM HEPES buffer (pH 7.4), 10 mM halogen donor, 0.5 mM hydroxylated phenazine, 100 μM FAD, 5 μM PezW protein, 1 mM NADH, 27 μL ddH2O; placed in a 30°C water bath for 4 hours.

[0024] Beneficial effects of the present invention:

[0025] (1) The present invention provides a phenazine halogenase PezW, which realizes the enzymatic halogenation of hydroxylated phenazines, fills the technical gap in the enzymatic preparation of hydroxylated phenazines, and produces significant technical effects.

[0026] (2) The phenazine halogenase PezW described in this application realizes the catalytic conversion of halogen atoms (Cl - ,Br - ,I - ) to hydroxylated phenazine substrates, which is of milestone significance for the development of enzymatic synthesis of HHPs technology.

[0027] (3) The phenazine halogenase PezW described in the present invention can halogenate hydroxylated phenazines through an enzyme-catalyzed reaction, providing a new path for the development of drugs for phenazine compounds, and has important application value and social significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Attachment Figure 1 This is an electrophoretogram of SDS-PAGE analysis of the phenazine halogenase PezW purified in Example 1 of the present invention;

[0029] Attachment Figure 2 This is a high performance liquid chromatography (HPLC) chart of the system after the reaction of phenazine halogenase PezW with 1-hydroxyphenazine in Example 2-4 of the present invention;

[0030] Attachment Figure 3 This is a high performance liquid chromatography (HPLC) chart of the system after the reaction of phenazine halogenase PezW with 1,6-dihydroxyphenazine in Examples 5-7 of the present invention;

[0031] Attachment Figure 4 This is the UV absorption graph of the substrate and product of the reaction between phenazine halogenase PezW and 1-hydroxyphenazine in Example 2-4 of the present invention;

[0032] Attachment Figure 5 This is the UV absorption graph of the substrate and product of the reaction between phenazine halogenase PezW and 1,6-dihydroxyphenazine in Examples 5-7 of the present invention;

[0033] Attachment Figure 6This is a high-resolution mass spectrometry (HR-MS) spectrum of the reaction product of phenazine halogenase PezW and 1-hydroxyphenazine in the presence of NaCl as a halogen donor in Example 2 of the present invention;

[0034] Attachment Figure 7 This is a high-resolution mass spectrometry (HR-MS) spectrum of the reaction product of phenazine halogenase PezW and 1-hydroxyphenazine in the presence of NaBr as a halogen donor in Example 3 of the present invention;

[0035] Attachment Figure 8 This is a high-resolution mass spectrometry (HR-MS) spectrum of the reaction product of phenazine halogenase PezW and 1-hydroxyphenazine in the presence of NaI as a halogen donor in Example 4 of the present invention;

[0036] Attachment Figure 9 This is a high-resolution mass spectrometry (HR-MS) spectrum of the reaction product of phenazine halogenase PezW and 1,6-dihydroxyphenazine in the presence of NaCl as a halogen donor in Example 5 of the present invention;

[0037] Attachment Figure 10 This is a high-resolution mass spectrometry (HR-MS) spectrum of the reaction product of phenazine halogenase PezW and 1,6-dihydroxyphenazine in the presence of NaBr as a halogen donor in Example 6 of the present invention;

[0038] Attachment Figure 11 This is a high-resolution mass spectrometry (HR-MS) spectrum of the reaction product of phenazine halogenase PezW and 1,6-dihydroxyphenazine in the presence of NaI as a halogen donor in Example 7 of the present invention.

[0039] Attachment Figure 12 The structural formula of the hydroxylated phenazine and the halogenated product in the halogenation reaction of Example 2-7 is shown. DETAILED DESCRIPTION

[0040] The present invention will be further described below with reference to the embodiments.

[0041] The raw materials and chemical reagents used in the examples of this application were all purchased from commercial sources. HEPES buffer was purchased from Shanghai MacLean Biological Reagent Co., Ltd.; NaCl, NaBr, and NaI were purchased from Shanghai Sangon Biotechnology Co., Ltd.; FAD (Flavin Adenine Dinucleotide) was purchased from Sigma-Aldrich; and NADH (Nicotinamide Adenine Dinucleotide) was purchased from Sigma-Aldrich.

[0042] Example 1: Cloning and in vitro expression of the halogenase PezW gene

[0043] 1. Synthesis of PezW gene

[0044] The nucleotide sequence of PezW (SEQ ID NO: 2) was synthesized by Qingke Biotechnology Co., Ltd. and ligated into the pUC19 vector. The synthesized vector dry powder was dissolved in an appropriate amount of water and set aside.

[0045] 2. Construction of protein expression vector

[0046] PCR was performed using the water-soluble vector as a template.

[0047] Design primer pairs:

[0048] P1: 5'- ctcgag gccgaagaggctcttgatgcgc-3' / P2:5'- catatg atggataacgaactgcgggacgac-3'

[0049] PCR reaction system:

[0050] 5 μL (50 pmol) of each primer pair P1 and P2, 2 μL of template, 50 μL (0.5 U / μL) of 2× DNA polymerase, and distilled water were added to 100 μL.

[0051] PCR conditions:

[0052] Promoter amplification conditions: denaturation at 95°C for 5 min; 28 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s; 72°C for 5 min; functional gene amplification conditions: denaturation at 95°C for 5 min; 28 cycles of 95°C for 30 s, 65°C for 30 s, and 72°C for 1 min 30 s; 72°C for 5 min.

[0053] The amplified DNA fragment was digested with NdeI and XhoI restriction endonucleases, respectively, and cloned into the plasmid pET-30a to construct the plasmid pET-30a-PezW. The fragment was then introduced into Escherichia coli BL21(DE3) cells and induced with isopropylthiogalactoside (IPTG) for expression of the target protein. The cells were cultured at 16°C for 16 hours, harvested by centrifugation, resolubilized in a buffer (0.05M Tris-HCl, 0.5M NaCl, pH 7.5), and disrupted by ultrafiltration. The enzyme extract was obtained after affinity purification using a Ni column and concentration by ultrafiltration.

[0054] The enzyme extract was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Figure 1 .Depend on Figure 1 It can be seen that the protein prepared in this example is PezW with a molecular weight of 59.9 kDa.

[0055] The aforementioned enzyme extract was concentrated, replaced with a buffer solution (0.025 M Tris-HCl, 0.02 M NaCl, 10% glycerol, pH 7.5), and stored at -80°C until use.

[0056] Example 2: In vitro enzyme activity detection of PezW protein

[0057] (1) Prepare in vitro enzyme activity reaction system (100 μL):

[0058] 50 mM HEPES buffer (pH 7.4): 50 μL; 10 mM NaCl: 10 μL; 0.5 mM 1-hydroxyphenazine: 3 μL; 100 μM FAD: 2 μL; 5 μM PezW protein (prepared in Example 1): 5 μL; 1 mM NADH: 3 μL; distilled water: 27 μL.

[0059] (2) Halogenation reaction:

[0060] The enzyme activity reaction system prepared in step (1) was reacted at 30°C for 4 hours. After the reaction, 100 μL of methanol was added, vortexed for 5 minutes, and centrifuged at 13,000 rpm for 20 minutes. The precipitate was discarded to obtain the supernatant, which was the sample for in vitro enzyme activity detection and HR-MS detection.

[0061] Example 3: In vitro enzyme activity detection of PezW protein

[0062] The difference from Example 2 is that

[0063] (1) Prepare in vitro enzyme activity reaction system (100 μL):

[0064] 50 mM HEPES buffer (pH 7.4): 50 μL; 10 mM NaBr: 10 μL; 0.5 mM 1-hydroxyphenazine: 3 μL; 100 μM FAD: 2 μL; 5 μM PezW protein: 5 μL; 1 mM NADH: 3 μL; distilled water: 27 μL

[0065] (2) Halogenation reaction: Same as Example 2.

[0066] Example 4: In vitro enzyme activity detection of PezW protein

[0067] The difference from Example 2 is that

[0068] (1) Prepare in vitro enzyme activity reaction system (100 μL):

[0069] 50 mM HEPES buffer (pH 7.4): 50 μL; 10 mM NaI: 10 μL; 0.5 mM 1-hydroxyphenazine: 3 μL; 100 μM FAD: 2 μL; 5 μM PezW protein: 5 μL; 1 mM NADH: 3 μL; distilled water: 27 μL

[0070] (2) Halogenation reaction: Same as Example 2.

[0071] Example 5: In vitro enzyme activity detection of PezW protein

[0072] The difference from Example 2 is that

[0073] (1) Prepare in vitro enzyme activity reaction system (100 μL):

[0074] 50 mM HEPES buffer (pH 7.4): 50 μL; 10 mM NaCl: 10 μL; 0.5 mM 1,6-dihydroxyphenazine: 3 μL; 100 μM FAD: 2 μL; 5 μM PezW protein: 5 μL; 1 mM NADH: 3 μL; distilled water: 27 μL

[0075] (2) Halogenation reaction: Same as Example 2.

[0076] Example 6: In vitro enzyme activity detection of PezW protein

[0077] The difference from Example 2 is that

[0078] (1) Prepare in vitro enzyme activity reaction system (100 μL):

[0079] 50 mM HEPES buffer (pH 7.4): 50 μL; 10 mM NaBr: 10 μL; 0.5 mM 1,6-dihydroxyphenazine: 3 μL; 100 μM FAD: 2 μL; 5 μM PezW protein: 5 μL; 1 mM NADH: 3 μL; distilled water: 27 μL

[0080] (2) Halogenation reaction: Same as Example 2.

[0081] Example 7: In vitro enzyme activity detection of PezW protein

[0082] The difference from Example 2 is that

[0083] (1) Prepare in vitro enzyme activity reaction system (100 μL):

[0084] 50 mM HEPES buffer (pH 7.4): 50 μL; 10 mM NaI: 10 μL; 0.5 mM 1,6-dihydroxyphenazine: 3 μL; 100 μM FAD: 2 μL; 5 μM PezW protein: 5 μL; 1 mM NADH: 3 μL; distilled water: 27 μL

[0085] (2) Halogenation reaction: Same as Example 2.

[0086] Example 8: HPLC analysis of the halogenation reaction products in Examples 2-7

[0087] HPLC detection conditions: Use a reverse-phase C18 column (specifications: 150×4.6 mm, 5 μm); column temperature, 30°C; elution conditions: 0-5 min equilibrium: 70% phase A (distilled water + 0.1% formic acid) and 30% phase B (acetonitrile + 0.1% formic acid); 5-45 min linear elution, 70-0% phase A and 30-100% phase B; 45-50 min isocratic elution: phase A: 0%, phase B: 100%; detection wavelength, 280 nm; flow rate, 1 mL / min.

[0088] Attachment Figure 2 The HPLC results of the system after the reaction of phenazine halogenase PezW and 1-hydroxyphenazine in Example 2-4 are shown. Figure 2 It can be seen that the HPLC spectrum of the sample containing inactivated PezW and 1-hydroxyphenazine only has a chromatographic peak of the substrate 1-hydroxyphenazine at a retention time of 16 minutes. However, the HPLC spectrum of the sample containing PezW + 1-hydroxyphenazine + a halogen donor shows new chromatographic peaks. Among them, when the halogen donor is NaCl, three chromatographic peaks appear at retention times of 19.6 minutes, 20.7 minutes, and 21.2 minutes. When the halogen donor is NaBr, three chromatographic peaks appear at retention times of 21.2 minutes, 21.4 minutes, and 22 minutes. When the halogen donor is NaI, a chromatographic peak appears at a retention time of 23.7 minutes.

[0089] Attachment Figure 3 The HPLC results of the system after the reaction of phenazine halogenase PezW and 1,6-dihydroxyphenazine in Example 5-7 are shown. Figure 3It can be seen that the HPLC spectrum of the sample containing inactivated PezW and 1,6-dihydroxyphenazine only shows the chromatographic peak of the substrate 1,6-dihydroxyphenazine at a retention time of 17.8 minutes. However, the HPLC spectrum of the sample containing PezW + 1,6-dihydroxyphenazine + a halogen donor shows new chromatographic peaks. Among them, when the halogen donor is NaCl, four chromatographic peaks appear at retention times of 22.1 minutes, 22.3 minutes, 25.5 minutes, and 26.0 minutes. When the halogen donor is NaBr, three chromatographic peaks appear at retention times of 23.2 minutes, 23.5 minutes, and 26.5 minutes. When the halogen donor is NaI, a chromatographic peak appears at a retention time of 25.0 minutes.

[0090] In summary, according to the HPLC results, different peaks were generated in the systems using NaCl, NaBr, and NaI as halogen donors and 1-hydroxyphenazine and 1,6-dihydroxyphenazine as substrates. This indicates that the hydroxylated phenazine reacted chemically with the phenazine halogenase PezW to produce different products.

[0091] Example 9: UV Detection Analysis of Halogenation Reaction Products in Examples 2-7

[0092] UV detection conditions: Diode Array Detector (DAD), detection range: 190-400 nm.

[0093] Attachment Figure 4 The UV absorption graph of the substrate and product of the reaction between phenazine halogenase PezW and 1-hydroxyphenazine in Example 2-4. Figure 4 It can be seen that the UV absorption peak of compound 1 (1-hydroxyphenazine) is located at 270 nm, and the UV absorption peaks of compounds 1a-1g are also located at 270 nm, which indicates that compounds 1a-1g are halogenated products of compound 1.

[0094] Attachment Figure 5 This is the UV absorption graph of the substrate and product of the reaction between phenazine halogenase PezW and 1,6-dihydroxyphenazine in Example 5-7 of the present invention. Figure 5 It can be seen that the UV absorption peak of compound 2 (1,6-dihydroxyphenazine) is located at 280 nm, and the UV absorption peaks of compounds 2a-2h are also located at 280 nm. This indicates that compounds 2a-2h are halogenated products of compound 2.

[0095] Combined with the HPLC spectra of Example 8 ( Figure 2-3) It can be seen that the reaction of 1-hydroxyphenazine / 1,6-dihydroxyphenazine with phenazine halogenase PezW produces a halogenated product, which indicates the occurrence of an enzymatic reaction; thereby confirming that the halogenase PezW provided in the present application achieves the catalytic transfer of halogen atoms to hydroxylated phenazine substrates.

[0096] Example 10: High Resolution Mass Spectrometry (HR-MS) Analysis of Halogenation Reaction Products in Examples 2-7

[0097] HR-MS analysis conditions: use a reversed-phase C18 column (specifications: 150×4.6 mm, 5 μm); column temperature is 30°C; elution conditions: 0-5 min equilibrium: 95% phase A (distilled water + 0.1% formic acid) and 5% phase B (acetonitrile + 0.1% formic acid); 5-15 min linear elution, 95-5% phase A and 5-95% phase B; 15-18 min isocratic elution: phase A: 0%, phase B: 100%; detection wavelength is 280 nm; flow rate is 0.3 mL / min; ion source type is: electrospray ionization (ESI); ion mode: positive ion mode; acquisition range: m / z: 200-1000 Da.

[0098] Attachment Figure 6 HR-MS spectra of the reaction products 1a-1c of phenazine halogenase PezW and 1-hydroxyphenazine (NaCl as halogen donor) in Example 2. Figure 6 When NaCl was used as the halogen donor and the substrate was 1-hydroxyphenazine (1, [M+H] = 197.07), the [M+H] values ​​of products 1a-1b were 231.03, and 1c was 264.99. Compared to the substrate molecular weight, the molecular weights of 1a-1b increased by 33.96 Da, confirming that they were monochlorinated products; while the molecular weight of 1c increased by 67.92 Da, confirming that it was a dichlorinated product. These molecular weights were consistent with those of the expected target product, indicating that chlorination occurred.

[0099] Attachment Figure 7 1d-1f are HR-MS spectra of the reaction products 1d-1f of phenazine halogenase PezW and 1-hydroxyphenazine (NaBr as halogen donor) in Example 3. Figure 7When NaBr was used as the halogen donor and the substrate was 1-hydroxyphenazine (1, [M+H] = 197.07), the [M+H] values ​​of products 1d-1e and 1f were 274.98 and 354.89, respectively. Compared to the substrate, the molecular weights of 1d-1e increased by 77.91 Da, confirming that they were monobrominated products; while the molecular weight of 1f increased by 155.82 Da, confirming that 1f was dibrominated. These molecular weights were consistent with those of the expected target product, indicating that bromination occurred.

[0100] Attachment Figure 8 HR-MS spectrum of the reaction product 1g of phenazine halogenase PezW and 1-hydroxyphenazine (NaI is the halogen donor) in Example 4. Figure 8 When NaI was used as the halogen donor and the substrate was 1-hydroxyphenazine (1, [M+H] = 197.07), the [M+H] of the product 1g was 322.97. Compared to the substrate molecular weight, the molecular weight of 1g increased by 125.90 Da, confirming that 1g is a monoiodinated product. The molecular weight is consistent with that of the expected target product, indicating that the iodination reaction occurred.

[0101] Attachment Figure 9 HR-MS spectra of the reaction products 2a-2d of phenazine halogenase PezW and 1,6-dihydroxyphenazine (NaCl as halogen donor) in Example 5. Figure 9 When NaCl was used as the halogen donor and the substrate was 1,6-dihydroxyphenazine (2, [M+H] = 213.07), the [M+H] values ​​of products 2a-2b were 247.02, and those of 2c-2d were 280.99. Compared to the substrate molecular weight, the molecular weights of 2a-2b increased by 33.96 Da, confirming that 2a-2b were monochlorinated products; while the molecular weights of 2c-2d increased by 67.92 Da, confirming that 2c-2d were dichlorinated products. These molecular weights were consistent with those of the expected target products, indicating that chlorination occurred.

[0102] Attachment Figure 10 HR-MS spectrum of the reaction product 2e-2g of phenazine halogenase PezW and 1,6-dihydroxyphenazine (NaBr as halogen donor) in Example 6. Figure 10When NaBr was used as the halogen donor and the substrate was 1,6-dihydroxyphenazine (2, [M+H] = 213.07), the [M+H] values ​​of the products 2e-2f and 2g were 290.98 and 370.88, respectively. Compared to the substrate molecular weight, the molecular weight of 2e-2f increased by 77.91 Da, confirming that 2e-2f is a monobrominated product; the molecular weight of 2g increased by 155.82 Da, confirming that 2g is a dibrominated product. These molecular weights are consistent with those of the expected target product, indicating that bromination occurred.

[0103] Attachment Figure 11 HR-MS spectrum of the reaction product 2h of phenazine halogenase PezW and 1,6-dihydroxyphenazine (NaI as halogen donor) in Example 7. Figure 11 When NaI was used as the halogen donor and the substrate was 1,6-dihydroxyphenazine (2, [M+H] = 213.07), the product 2h had a [M+H] of 338.96. Compared to the substrate, the molecular weight of 2h increased by 125.90 Da, confirming that 1g was a monoiodinated product. This molecular weight was consistent with that of the expected target product, indicating that the iodination reaction occurred.

[0104] In summary, the halogenase PezW provided in this application achieves the transfer of various halogen atoms from NaCl / NaBr / NaI to hydroxyphenazines, enabling the enzymatic halogenation of hydroxyphenazines. This fills a technological gap in the enzymatic preparation of hydroxyphenazines and yields significant technical benefits. This not only clears the way for the enzymatic preparation of various HHPs but also provides a new path for the development of phenazine-based pharmaceuticals, possessing significant social significance and promising applications.

Claims

1. A phenazine halogenase PezW, characterized in that: The amino acid sequence of the phenazine halogenase PezW is selected from the following (1), (2) or (3): (1) the amino acid sequence shown in SEQ ID NO: 1; (2) an amino acid sequence as shown in SEQ ID NO: 1, wherein one or more amino acids are substituted, deleted, or added and the amino acid sequence is capable of catalyzing the transfer of a halogen atom to a hydroxylated phenazine; (3) An amino acid sequence having a homology of ≥90% with the amino acid sequence shown in SEQ ID NO: 1, and the expressed protein is capable of catalyzing the transfer of a halogen atom to a hydroxylated phenazine.

2. The DNA molecule encoding the phenazine halogenase PezW according to claim 1, characterized in that: The nucleotide sequence is selected from the following (1) or (2): (1) the nucleotide sequence shown in SEQ ID NO: 2; (2) a nucleotide sequence that is different from the nucleotide sequence shown in SEQ ID NO: 2 but encodes the amino acid sequence shown in SEQ ID NO: 1; (3) a nucleotide sequence that has a homology of ≥85% with the nucleotide sequence shown in SEQ ID NO: 2, and the protein expressed by the nucleotide sequence can catalyze the transfer of a halogen atom to a hydroxylated phenazine; (4) A nucleotide sequence complementary to the nucleotide sequence described in any one of (1), (2) or (3).

3. An isolated recombinant vector, characterized in that: An expression vector containing the nucleotide sequence encoding the phenazine halogenase PezW according to claim 2.

4. A host cell, characterized in that: Comprising the isolated recombinant vector as claimed in claim 3; the host cell does not include animal or plant species.

5. The method for cloning and expressing the phenazine halogenase PezW according to claim 1 or 2, comprising the following steps: cloning the nucleotide sequence encoding the phenazine halogenase PezW into an expression vector to construct an expression vector; then transferring the expression vector into an expression system for expression; and finally purifying to obtain the phenazine halogenase PezW.

6. Use of the phenazine halogenase PezW according to claim 1 or 2 in the preparation of halogenated compounds.

7. The use according to claim 6, characterized in that: The compound is a hydroxylated phenazine, specifically 1-hydroxyphenazine, 2-hydroxyphenazine or 1,6-dihydroxyphenazine.

8. The use according to claim 6, characterized in that: The halogenation modification is: transferring the halogen atom of the halogen donor to the phenazine compound and combining it; the halogen donor is NaCl, NaBr or NaI.

9. The use according to any one of claims 6 to 8, characterized in that: The halogenation modification is: halogenation modification of the ortho-position and para-position of the hydroxyl group in the hydroxylated phenazine is carried out through an enzyme-catalyzed reaction.

10. The use according to claim 9, characterized in that: The reaction system of the enzyme-catalyzed reaction is: 50 mM HEPES buffer (pH 7.4), 10 mM halogen donor, 0.5 mM hydroxylated phenazine, 100 μM FAD, 5 μM PezW protein, 1 mM NADH, and 27 μL distilled water.

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