Application of a siderophore derived from Lysobacterium

Abstract

The present invention discloses an application of a siderophore derived from Lysobacterium. High-purity siderophore was obtained from the fermentation broth of Lysobacter OH11 through fermentation culture, XAD16 resin adsorption and desorption, and HPLC preparation. Mass spectrometry and nuclear magnetic resonance confirmed that the siderophore was a catechol type, composed of two catechol groups and one spermidine, with a molecular formula of C 21 O6N3H 27 The molecular weight is 417Da and it is named Sperbactin. The plate antagonism test showed that Sperbactin has strong antagonistic activity against rice bacterial leaf blight and rice bacterial leaf streak pathogen. 50 The concentrations of these substances are 15.44 mg / L and 16.76 mg / L respectively, which can be developed into highly effective bactericides.

Description

[0001] This application is a divisional application of the Chinese invention patent with the application date of September 14, 2022, application number 202211118311.9, and the invention name is "A siderophore derived from lysobacteria, its preparation method and application". Technical Field

[0002] The present invention belongs to the field of biotechnology and specifically relates to an application of a siderophore derived from Lysobacterium background

[0003] Lysobacter, belonging to the Xanthomonadaceae family and the genus Lysobacter, is an important, yet underexplored, class of plant disease biocontrol bacteria, found widely in soil and freshwater environments. As of 2019, 50 species of Lysobacter have been reported internationally. Among them, Lysobacter enzymogenes is a typical species of the genus and one of the most studied biocontrol bacteria. This bacterium produces abundant extracellular hydrolases and small molecule antimicrobial products, demonstrating significant antagonistic effects against important crop pathogens, including fungi, oomycetes, bacteria, and nematodes.

[0004] Siderophores (also called iron carriers) are secondary metabolites synthesized and secreted extracellularly by bacteria and fungi in iron-deficient environments. They are essentially non-protein amino acids with a relative molecular mass of less than 10 kDa. They have a strong affinity for trivalent iron and increase iron solubility by lowering the environmental pH. To date, there are no reports on the application of siderophores in Lysobacter. Therefore, isolating and identifying siderophores from Lysobacter and studying their applications would not only enrich the types and functions of Lysobacter active products and help to elucidate the mechanism of action of Lysobacter, but also provide a material basis for the development of new biocontrol agents. Summary of the Invention

[0005] The present invention aims to provide an application of siderophore derived from lysobacterium.

[0006] To achieve the above objectives, the technical solutions adopted by the present invention are:

[0007] In a first aspect, the present invention provides a siderophore isolated and purified from the fermentation broth of Bacillus enzymogenes OH11. Furthermore, the chemical structure of the siderophore is identified as follows:

[0008]

[0009] The siderophore described in the present invention is of catechol type, has a molecular weight of 417 Da, is composed of two catechol groups and one spermidine, and is named Sperbactin.

[0010] In a second aspect, the present invention further provides a method for preparing the siderophore described in the first aspect, comprising the following steps: (1) preparing a fermentation broth of Bacillus enzymolyticus OH11; (2) preparing a siderophore desorption solution; and (3) isolating and purifying the siderophore.

[0011] The Lysobacter enzymogenes OH11 described in the present invention is a strain independently isolated by our laboratory and deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms on March 19, 2007, with the culture collection number CGMCC NO.1978 (see patent ZL200710190998.6).

[0012] In some embodiments, step (1) of the present invention is to inoculate the enzyme-producing Bacillus lysozyme OH11 into a fermentation medium for fermentation. In some specific examples, the fermentation process is: the activated strain OH11 is inoculated into LB liquid culture medium and cultured at 26-28°C and 170-190 rpm for 12-15 hours, and then transferred to the fermentation medium at an inoculum size of 1.0-2.5%, and cultured at 26-29°C and 150-200 rpm for 48-60 hours.

[0013] In some embodiments, the fermentation medium of the present invention comprises: glucose 5-15 g / L, (NH4)2SO4 0.5-2.0 g / L, MgCl2 5-20 mg / L, K2HPO4 0.5-4.0 g / L, KH2PO4 0.25-2.0 g / L, and CaCO3 0.25-1.5 g / L. Preferably, the fermentation medium comprises: glucose 5-10 g / L, (NH4)2SO4 0.8-1.2 g / L, MgCl2 8-15 mg / L, K2HPO4 1.0-1.5 g / L, KH2PO4 0.5-0.75 g / L, and CaCO3 0.5-1.0 g / L. More preferably, the fermentation medium comprises 8 g / L glucose, 1.0 g / L (NH₄)₂SO₄, 10 mg / L MgCl₂, 1.0 g / L K₂HPO₄, 0.5 g / L KH₂PO₄, and 1.0 g / L CaCO₃. The fermentation medium is prepared with pure water. The fermentation medium of the present invention can effectively increase the production of siderophore, reaching a level of 100-220 mg / L.

[0014] In some embodiments, step (2) of the present invention specifically comprises directly mixing the fermentation broth obtained in step (1) with XAD16 resin, placing the mixture on a shaker for adsorption for 12 to 24 hours, filtering the resin and the filtrate, and desorbing the resin using anhydrous ethanol to obtain a desorbed liquid. The XAD16 resin of the present invention can be purchased commercially.

[0015] In some embodiments, the adsorption conditions of the present invention are: resin addition amount is 20-40 g / L, shaker 120-200 rpm, 26-30°C;

[0016] In some embodiments, the desorption conditions described herein are: anhydrous ethanol to macroporous resin volume mass ratio of 20-50 mL:1 g (wet weight), preferably 25 mL:1 g. The desorption conditions are shaker at 120-200 rpm, 27-37° C., for 0.5-3 hours. In a specific embodiment, the shaker is at 180 rpm, 28° C., for 2 hours.

[0017] In some embodiments, step (3) of the present invention is specifically to rotary evaporate the desorption liquid obtained in step (2), redissolve it in methanol solution, separate and purify it by preparative HPLC, collect the siderophore fraction, rotary evaporate it, and freeze-dry it to obtain pure siderophore.

[0018] In some embodiments, the HPLC preparation conditions of the present invention are: InterSustainSwift C18 5μm, 250×20mm; mobile phase: solution A (0.025% TFA aqueous solution) and solution B (0.025% TFA acetonitrile solution), flow rate: 1-2 mL / min; injection volume: 3 mL, UV absorbance: 254 nm; injection program: 0-10 min, increase solution B from 5% to 25%; 25 min, increase to 80% B; 26 min, increase to 100%; 27-30 min return to 5%, the total ratio of solution A and B in the whole process is 100%; the fraction collection time is 11.7-12.2 min.

[0019] In a third aspect, the present invention further provides use of the siderophore described in the first aspect in preparing a biocontrol agent.

[0020] The present invention also provides use of the siderophore described in the first aspect as an iron carrier.

[0021] The present invention also provides use of the siderophore described in the first aspect in preventing and controlling plant pathogenic bacteria.

[0022] The bacteria described in the present invention are Xanthomonas oryzae pv.oryzae PXO99, Xanthomonas oryzae pv.oryzicola Rs105, Pseudomonas syringae, and Pseudomonas syringae pv.Lachrymans. Further, the bacteria are Xanthomonas oryzae pv.oryzae PXO99 or Xanthomonas oryzae pv.oryzicola Rs105, EC 50 They are 15.44mg / L and 16.76mg / L respectively.

[0023] The method of the present invention has the following advantages over the prior art:

[0024] (1) There are currently no reports on the production of siderophores from Lysobacter. The siderophore described in the present invention is a new active compound isolated and identified from Lysobacter, which can be developed into a new biocontrol agent or antibacterial drug. It can also provide iron nutrients for plant growth and has important application value in the field of biological control.

[0025] (2) The siderophore described in the present invention has a strong antagonistic effect on rice bacterial leaf blight and rice bacterial leaf streak pathogen, and can be developed into a specific fungicide for the prevention and control of rice bacterial diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 CAS plate test;

[0027] Figure 2 Arnow reaction of fermentation broth supernatant;

[0028] Figure 3 Arnow reaction of filtrate and desorbate;

[0029] Figure 4 HPLC analysis of desorption liquid components;

[0030] Figure 5 Mass spectrometry analysis;

[0031] Figure 6 NMR analysis;

[0032] Figure 7 Standard curve for the quantitative analysis of siderophore;

[0033] Figure 8 EC of siderophores to pathogens 50Determination. DETAILED DESCRIPTION

[0034] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

[0035] Example 1: Detection of the ability of Lysobacterium enzymogenes OH11 to produce siderophore

[0036] Siderophore detection dye solution: Dissolve 0.079g CAS in 50mL deionized water, then add 10mL of 1mmol / LFeCl3 solution (containing 12mmol / L HCl) to obtain Solution A. Dissolve 0.069g hexadecyltrimethylammonium bromide (HDTMA) in 40mL deionized water to obtain Solution B. Slowly add Solution A to Solution B along the side of the beaker and stir to mix to obtain 100mL of CAS blue detection solution.

[0037] CAS medium: Each 1L contains 10mL of 20% sucrose solution, 30mL of 10% acid hydrolyzed casein, 1000μL of 1mmol / L CaCl2, 20mL of 1mmol / L MgSO4, and 18g of agar. Slowly add 50mL each of phosphate buffer and CAS detection dye at about 60℃;

[0038] A single colony of Bacillus lysozyme-producing OH11 was streaked from an LB solid plate into 50 mL of LB culture medium and cultured at 28°C with shaking at 180 rpm for 12 hours to obtain a bacterial suspension. 3 μL of the bacterial suspension was spotted around the CAS solid plate and cultured in a 28°C incubator for 2 days. Figure 1 As shown, an orange halo was observed around the colonies, indicating that the OH11 strain had the ability to produce siderophore.

[0039] Example 2: Determination of the siderophore type produced by Lysobacterium enzymogenes OH11

[0040] The Arnow method for detecting catechol-type siderophores is as follows: 0.5M HCl; 10g sodium nitrite and 10g sodium molybdate dissolved in a small amount of water and then made up to 100mL; 1M NaOH.

[0041] Fermentation medium formulation: 5 g / L glucose, 2 g / L (NH₄)₂SO₄, 1.5 g / L K₂HPO₄, and 0.75 g / L KH₂PO₄. Prepare 1 L of this medium with purified water, aliquot 50 mL into 250 mL Erlenmeyer flasks, and sterilize at 121°C for 20 min. A control medium containing 8 mg / L FeCl₃ was also added. Seed solution preparation was the same as in Example 1. A 2.5% inoculum was inoculated into the fermentation medium and cultured at 28°C and 180 rpm for 48 h to obtain a fermentation broth.

[0042] The fermentation broth was centrifuged at 10000 rpm for 5 min, and 1 mL of supernatant was mixed with 1 mL of 0.5 M HCl, 1 mL of molybdate-sodium nitrite, and 1 mL of NaOH. If catechol-type siderophores are present, the color of the mixture will turn red. Figure 2 As shown, without adding Fe 3+ There was an obvious red reaction in the fermentation broth, indicating that the siderophore produced by OH11 was of catechol type.

[0043] Example 3: Preparation of Siderophore Desorption Solution

[0044] 2 g (wet weight) of pretreated XAD16 resin was added to the fermentation broth of Example 2, and the mixture was placed in a shaker at 28°C and 180 rpm for overnight shaking to achieve full adsorption. The resin and the filtrate were separated by 3 layers of gauze, and the filtrate was subjected to an Arnow reaction to detect the siderophore content. The adsorbed resin was desorbed by adding 50 mL of anhydrous ethanol, placing the mixture in a shaker at 28°C and 180 rpm for 2 h. The desorbed liquid was collected and subjected to an Arnow reaction. The mixture was concentrated on a rotary evaporator at 50°C, evaporated to dryness, dissolved in methanol, and filtered through a 0.22 μm filter membrane for HPLC analysis. The injection volume was 20 μL, and the detection conditions were as follows: UV absorption value: 254 nm, reversed-phase column: InterSustainSwift C18 5 μm, 250×4.6 mm; mobile phase: solution A (0.025% TFA aqueous solution) and solution B (0.025% TFA acetonitrile solution), flow rate: 1 mL / min; injection program: 0-10 min, solution B increased from 5% to 25%; 25 min, increased to 80% B; 26 min, increased to 100%; 30 min returned to 5%, and the total ratio of solution A and B in the whole process was 100%.

[0045] Depend on Figure 3 It can be seen that the filtrate contains almost no siderophore, while the desorption liquid shows an obvious red color, indicating that the siderophore in the fermentation broth can be transferred into the desorption liquid through resin adsorption. Figure 4 It can be seen that compared with the control, the 3+The desorbed liquid has an obvious absorption peak on the HPLC chromatogram with a retention time of 11.9 min. This peak is the absorption peak of catechol-type siderophore produced by OH11.

[0046] Example 4: Purification and identification of siderophores

[0047] The siderophore desorbate from Example 3 was further separated and purified using HPLC. The fraction with a retention time of 11.7-12.2 min was collected and freeze-dried for further structural identification. Preparation conditions included an InterSustainSwift C18 5μm, 250×20mm reversed-phase column, a flow rate of 5mL / min, and an injection volume of 3mL. Other analytical procedures were the same.

[0048] The siderophore fractions purified by HPLC were analyzed by mass spectrometry. Figure 5 As shown (HRMS (ESI): calculated for C 21 H 28 N3O6[M+H] + 418.1978, found 418.2000; calculated for C 21 H 26 N3O6[MH] - 416.1822, found 416.2000), the molecular weight of this component is 417Da. Figure 6 As shown ( 1 H NMR (400MHz, CD3OD): δ7.21(dd,1H,J=8.0,1.6Hz), 7.21(dd,1H,J=8.4,1.6Hz), 6.95(dd,1H,J=6.0,1.6Hz), 6.93(dd,1H,J=6.0,1.6Hz), 6.93(t,1 H,J=8.0Hz),6.72(t,1H,J=7.6Hz),3.52(t,2H,J=6.4Hz),3.46(t,2H,J=6.0Hz),3.11-3.04(m,4H),1.99(quint,2H,J=7.2Hz),1.82-1.70(m,4H).

[0049] 13C NMR (100 MHz, CD3OD): δ170.78, 170.30, 148.81, 148.64, 145.97, 145.95, 118.47, 118.43, 118.29, 118.27, 117.52, 117.29, 115.36, 115.25, 47.30, 45.16, 38.08, 35.71, 26.30, 26.17, 23.27). Based on the above analysis, the inferred siderophore structure is C 21 O6N3H 27 The chemical structure is shown below. It contains two catechol active groups and one spermidine structure. We named it Sperbactin.

[0050]

[0051] Example 5: Quantitative detection of siderophores in the fermentation broth of Lysobacter OH11

[0052] A standard curve between the siderophore concentration and the absorption peak area was established. The specific process was as follows: 10 mg of the siderophore pure product prepared in Example 4 was weighed and added to 10 mL of methanol solution to prepare a 1000 mg / L mother solution, and then diluted to different gradient concentrations in sequence. Finally, the solution was detected by HPLC column analysis under the same conditions as in Example 3. The peak area was recorded, and a standard curve was drawn with the siderophore concentration in the prepared solution as the ordinate (Y) and the absorption peak area as the abscissa (X). Figure 7 As shown in the figure, the linear equation between the siderophore concentration and the peak area is Y = 0.0483*X-7.0726, R 2 =0.9999.

[0053] Example 6: Fermentation medium for high-siderophore production of Lysobacterium enzymogenes OH11

[0054] The fermentation medium formula for high-yield siderophore is as follows: 8 g / L glucose, 1 g / L (NH4)2SO4, 10 mg / L MgCl2, 1 g / L K2HPO4, 0.5 g / L KH2PO4, and 1 g / L CaCO3. 1 L of the medium is prepared with pure water, and 50 mL is dispensed into 250 mL Erlenmeyer flasks and sterilized at 121°C for 20 min. The seed solution preparation method is the same as that of Example 2. The seed solution is inoculated into the fermentation medium at a 2.5% inoculum amount, and cultured at 28°C and 180 rpm for 48 h to obtain a fermentation broth. The results showed that the siderophore production of the OH11 strain in this medium reached 213.99 mg / L, while the siderophore production of Example 2 was 108.24 mg / L. It can be seen that the culture medium of Example 6 effectively increased the production of siderophore, laying the foundation for its further development.

[0055] Example 7: Application of siderophore

[0056] 10 mg of the pure siderophore obtained in Example 4 was accurately weighed and added to 1 mL of DMSO solution to prepare a 10 mg / mL stock solution.

[0057] The pathogens tested included Pseudomonas syringae pv. Lachrymans, Xanthomonas oryzae pv. oryzae PXO99, Xanthomonas oryzae pv. oryzicola Rs105, and Pseudomonas syringae. These pathogens are currently stored in this laboratory, and the inventors and applicants promise to make them permanently available to the public.

[0058] Pathogenic bacteria EC 50 Determination of pathogenic bacteria: culture the pathogenic bacteria in NB culture medium to OD 600 =0.5, mix according to the volume ratio of 1:1000, pipette 1mL of the mixture into a 24-well plate, and add the siderophore stock solution to make the final concentrations of 0μg / mL, 5μg / mL, 8μg / mL, 10μg / mL, 15μg / mL, 20μg / mL, 35μg / mL, 50μg / mL, 80μg / mL, and 100μg / mL respectively. Place in a shaker at 28℃, 180rpm and culture for 12-24h, and measure the OD 600 , and calculate EC 50 .

[0059] Depend on Figure 8 It can be seen that OH11 siderophore has a good inhibitory effect on Xanthomonas oryzae pv.oryzaePXO99, Xanthomonas oryzae pv.oryzicola Rs105, Pseudomonas syringae, and Pseudomonas syringae pv.Lachrymans, the bacterial leaf spot pathogen of rice. 50 The concentrations of these agents are 15.44 mg / L, 16.76 mg / L, 62.74 mg / L and 73.78 mg / L respectively. They are especially effective against Xanthomonas oryzae and Bacterial leaf stripe pathogen of rice. They can be developed into highly effective fungicides for the prevention and control of bacterial diseases of rice.

Claims

1. An application of siderophore in preventing and controlling plant pathogenic bacteria, characterized in that: The bacteria are one or more of Xanthomonas oryzae, Xanthomonas oryzae, Pseudomonas syringae, and Xanthomonas cucumeris. The chemical structure of the siderophore is as follows: 。 2. The use according to claim 1, characterized in that The bacteria are bacterial leaf blight pathogen or bacterial leaf streak pathogen.

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