Application of strain for producing benzohydroxamic acid
By screening and utilizing the natural benzohydroxamic acid in Streptocytica, the lack of biosynthesis of this compound was solved, and the effect of efficient biosynthesis and improving plant immunity was achieved, and a safe and environmentally friendly biopesticide was provided.
Patent Information
- Application Number
- CN202510230184.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-28
- Publication Date
- 2025-06-27
AI Technical Summary
The lack of methods for biosynthesis of benzohydroxamic acid in the prior art has led to its main chemical synthesis, and the application of biopesticides in plants to resist pathogenic fungi is limited.
By screening and determining the naturally occurring benzohydroxamic acid in Streptocytica, a method for producing benzohydroxamic acid is provided, and a method for preparing benzohydroxamic acid includes culturing the strain and increasing its yield by optimizing the culture medium, and finally obtaining the compound by extraction and identification.
It has achieved high yield biosynthesis of benzohydroxamic acid, providing a natural agent to improve plant immunity against pathogenic fungi, with safety and environmental protection advantages.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of microbial technology, and particularly relates to the application of a strain producing benzohydroxamic acid. Background Art
[0002] Plant fungal diseases are caused by pathogenic fungi infecting and parasitizing plants, accounting for 70% to 80% of all plant diseases, and almost all crops are affected. Due to the strong viability and wide spread of pathogenic fungi, the threat of plant fungal diseases to global agriculture is increasing. One way for plants to defend against pathogenic fungi is to recognize pathogens through MAMP (Microbe-Associated Molecular Patterns), and chitin is a key signaling molecule among them. However, pathogenic fungi can escape the activation of the plant immune system by secreting chitin deacetylase. Natural products are an important treasure trove for humans to develop drugs and materials.
[0003] Biopesticides are natural compounds extracted from organisms in nature or their metabolites, and have the advantages of being safe, environmentally friendly, and pollution-free. With the development of sustainable agriculture, biopesticides have become one of the key directions for future pesticide research and development due to their high efficiency and low toxicity. Due to the special structure of its hydroxamic acid group, benzohydroxamic acid has excellent metal chelating ability. Especially as an inhibitor of enzyme metal coenzymes, it can effectively inhibit chitin deacetylase in fungi, thereby enhancing the immunity of plants to pathogenic fungi. Currently, benzohydroxamic acid is mainly synthesized chemically, and there is no relevant report on biosynthesis. Summary of the Invention
[0004] Through research, the present invention determines that benzohydroxamic acid naturally exists in Streptomyces angustmyceticus, and thus provides an application of a strain producing benzohydroxamic acid in the production of benzohydroxamic acid. The Streptomyces angustmyceticus strain described in the present invention has a preservation number of CGMCC NO: 4.1918, and the classification name is Streptomyces angustmyceticus . It can be purchased.
[0005] The present invention also provides a method for preparing benzohydroxamic acid, which comprises the following steps: S1 Culturing the strain with a preservation number of CGMCC NO: 4.1918 to produce benzohydroxamic acid; optionally, further comprising: S2 Extracting the produced benzohydroxamic acid.
[0006] The specific step of S1 is: fermenting and culturing the strain in TSB medium or 2×YT medium to produce benzohydroxamic acid; The formula of the TSB medium is: tryptone 17 g, soy peptone 3 g, NaCl 5 g, glucose 2.5 g, K2HPO4 2.5 g, water 1000 ml.
[0007] The formula of the 2×YT medium is 16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride, and 1000 ml of water More specifically, the S1 step is as follows: Cultivate the strain in TSB medium on a shaker at 25-35°C and 160-300 rpm / min until small granular substances appear; take the bacterial liquid and add it to TSB medium or 2×YT medium, and then perform enlarged cultivation on a shaker at 25-35°C and 160-300 rpm / min until small granular substances appear.
[0008] Optionally, before the S1 step, pick a monoclonal strain from a Gao's No. 1 plate, verify it by 16sRNA, and then add it to TSB medium for cultivation; The formula of the Gao's No. 1 medium is: 20 g of starch, 0.5 g of NaCl, 1 g of KNO3, 0.5 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.01 g of FeSO4·7H2O, 15 g of agar, and 1000 ml of water.
[0009] This strain is derived from soil. On the Gao's No. 1 medium, it is cultured at 30°C for one week. The substrate mycelium develops well, aerial mycelium exists, the mycelium is white, and the bacterial cells are punctate.
[0010] The product extraction method described in the S2 step is: Use a vacuum freeze dryer to freeze-dry the obtained bacterial liquid until the moisture basically disappears, add methanol and use a vortex mixer to vortex, let it stand, perform ultrasonic treatment, centrifuge, and take the supernatant.
[0011] Specifically, vortex 5 times, perform ultrasonic treatment for 12-18 min, centrifuge at 8000-12000 rpm / min for 8-12 min, and take the supernatant.
[0012] Furthermore, it further includes filtering with a 0.22um nylon filter membrane to prepare a sample.
[0013] More specifically, the product extraction method is: Use a vacuum freeze dryer to freeze-dry 1 ml of bacterial liquid for 72 h until the moisture basically disappears, add 200 ul of methanol and use a vortex mixer to vortex for 1 min, let it stand for 1 min, and repeat vortexing 5 times. Perform ultrasonic treatment for 15 min, centrifuge at 10000 rpm / min for 10 min, and take the supernatant. Filter with a 0.22um nylon filter membrane to prepare a sample.
[0014] Further, the product identification method is as follows: Identification instrument: Agilent 8890-7010B GCMSMS gas chromatograph tandem triple quadrupole mass spectrometer. Chromatographic column: DB-HeavyWax (30.0m*0.25mm*0.25μm) capillary column. Heating program: initial temperature is 50°C, maintained for 2min; increased to 320°C at 15°C / min, maintained for 5min. Electron bombardment source: 70eV, EI ion source temperature: 280°C. Inlet temperature: 250°C, injection volume: 1.0μL, injection mode: 10:1 split injection. Agilent Qualitative Navigator B.08.00 was used for analysis and quantification.
[0015] Compared with the prior art, the present invention has the following beneficial effects: the present invention has discovered a strain of Streptomyces serrata that can produce benzohydroxamic acid in high yield through research and screening, and optimized the culture medium to further improve its yield. The present invention also further shows that benzohydroxamic acid naturally exists in Streptomyces serrata. In short, the strain of the present invention can be used to produce benzohydroxamic acid and can be used to prepare a medicament for improving the immunity of plants to pathogenic fungi. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.
[0017] Figure 1 :Extracted ion pattern of Streptomyces stenosis extract and benzohydroxamic acid standard by gas chromatography-mass spectrometry (m / z = 121.09), extraction time was 19.20–21.00 min.
[0018] Figure 2 : Mass spectrometric information of the chromatographic peak at 20.20min of benzohydroxamic acid standard and Streptomyces mycotoxins extract.
[0019] Figure 3 : Benzohydroxamic acid standard curve and quantification of production in TSB and 2×YT medium. Specific implementation methods
[0020] The instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.
[0021] Example 1: Biosynthesis of natural benzohydroxamic acid screening The present inventors screened a variety of bacteria to determine whether there were bacteria producing benzohydroxamic acid, and finally found that only Streptomyces angustmycinicus could produce benzohydroxamic acid. The detailed experimental procedures are provided below.
[0022] 1. Obtaining microorganisms and tobacco samples Pick a single colony of Escherichia coli BL21(DE3) from an LB plate, add the strain to 4 ml of LB liquid medium, and culture it in a shaker at 37 °C and 220 rpm / min for 16 h until it becomes completely turbid. Take 1 ml of the bacterial solution and add it to 50 ml of LB liquid medium, and culture it in a shaker at 37 °C and 220 rpm / min for 72 h, then take 1 ml of the bacterial solution.
[0023] Pick a single colony of Streptomyces angustmycinicus from a Gao's No. 1 plate, add the strain to 4 ml of TSB medium, and culture it in a shaker at 30 °C and 220 rpm / min for 48 h until small granular substances appear. Take 1 ml of the bacterial solution and add it to 50 ml of TSB medium, and culture it in a shaker at 30 °C and 220 rpm / min for 72 h, then take 1 ml of the bacterial solution.
[0024] Formulation of Gao's No. 1 medium: 20 g of starch, 0.5 g of NaCl, 1 g of KNO3, 0.5 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.01 g of FeSO4·7H2O, 15 g of agar, 1000 ml of water.
[0025] Formulation of TSB medium: 17 g of tryptone, 3 g of soy peptone, 5 g of NaCl, 2.5 g of glucose, 2.5 g of K2HPO4, 1000 ml of water.
[0026] Pick a single colony of Fusarium graminearum from a PDA plate, add the strain to 4 ml of PDA liquid medium, and culture it in a shaker at 30 °C and 220 rpm / min for 16 h until it becomes completely turbid. Take 1 ml of the bacterial solution and add it to 50 ml of PDA liquid medium, and culture it in a shaker at 30 °C and 220 rpm / min for 72 h, then take 1 ml of the bacterial solution.
[0027] Pick a single colony of Saccharomyces cerevisiae WAT11 from a YPDA plate, add the strain to 4 ml of YPD liquid medium, and culture it in a shaker at 30 °C and 220 rpm / min for 48 h until it becomes completely turbid. Take 1 ml of the bacterial solution and add it to 50 ml of YPD liquid medium, and culture it in a shaker at 30 °C and 220 rpm / min for 72 h, then take 1 ml of the bacterial solution.
[0028] Take fresh Nicotiana benthamiana leaves, freeze-dry them with liquid nitrogen and grind them into powder, and weigh 1 g of the powder.
[0029] 2. Product extraction 1 ml of bacterial solution was freeze-dried for 72 hours using a vacuum freeze dryer until the water was almost gone, 200 ul of methanol was added and vortexed for 1 minute using a vortexer, left to stand for 1 minute, and vortexed 5 times. Ultrasonication was performed for 15 minutes, and centrifugation was performed at 10000 rpm / min for 10 minutes, and the supernatant was collected. Filtered using a 0.22 um nylon filter membrane for sample preparation.
[0030] 1g tobacco leaf powder was freeze-dried in a vacuum freeze dryer for 72h until the water was almost gone, 200ul methanol was added and vortexed for 1min using a vortexer, left to stand for 1min, and vortexed 5 times. Ultrasonication was performed for 15min, and centrifugation was performed at 10000 rpm / min for 10min, and the supernatant was collected. Filtered using a 0.22um nylon filter membrane for sample preparation.
[0031] 3. Product identification Identification instrument: Agilent 8890-7010B GCMSMS gas chromatograph tandem triple quadrupole mass spectrometer. Chromatographic column: DB-HeavyWax (30.0m*0.25mm*0.25μm) capillary column. Heating program: initial temperature is 50℃, maintained for 2min; increase to 320℃ at 15℃ / min, maintained for 5min. Electron bombardment source: 70eV, EI ion source temperature: 280℃. Inlet temperature: 250℃, injection volume: 1.0μL, injection mode: 10:1 split injection. Agilent Qualitative NavigatorB.08.00 was used for analysis and quantification. The selected ion chromatogram was extracted using the benzohydroxamic acid characteristic ion 121.09, and the extraction time was 19.20–21.0 min. The results showed that except for Streptomyces stenosis, no chromatographic peaks similar to the chromatographic peak of benzohydroxamic acid standard were found in Escherichia coli, yeast and tobacco in the chromatographic results ( Figure 1 ). The mass spectrometry information was extracted from the chromatographic peak at 20.2 min of Streptomyces serrata and the benzohydroxamic acid standard. The results showed that the ion fragment information obtained was consistent with that of the benzohydroxamic acid standard ( Figure 2 ), quantitative analysis based on the chromatographic peak area showed that its content was approximately 0.05 mg / L.
[0032] Except for Streptomyces angustmycinicus, we tested whether other Actinobacteria contain benzohydroxamic acid. The above extraction and detection methods showed that most of them could not produce this substance. Only Saccharothrix syringae (CGMCC NO: 4.1716) also contained benzohydroxamic acid, and the quantitative amount through the standard curve was about 0.036 mg / L, with a lower yield than Streptomyces angustmycinicus. (Among them, due to the different other products produced by this Saccharothrix syringae strain, the detection method was modified to separate benzohydroxamic acid from other compounds in the matrix. The specific detection method is as follows: Identification instrument: Agilent 8890-7010B GCMSMS gas chromatography tandem triple quadrupole mass spectrometry. Chromatographic column: DB-HeavyWax (30.0m * 0.25mm * 0.25μm) capillary column. Temperature programming: The initial temperature is 50°C, held for 2 min; heated to 320°C at 15°C / min and held for 5 min. Electron impact source: 70 eV, EI ion source temperature: 280°C. Injection port temperature: 280°C, injection volume: 1.0 μL, injection mode: split injection at 20:1).
[0033] Example 2: Optimization of the strain fermentation medium 1 Microbial culture Pick a monoclonal strain of Streptomyces angustmycinicus from the Gao's No. 1 plate, add the strain to 4 ml of TSB medium, and culture it on a shaker at 30°C and 220 rpm / min for 48 h until small granular substances appear. Take 1 ml of the bacterial solution and add it to 50 ml of TSB and 2×YT media respectively, and culture it on a shaker at 30°C and 220 rpm / min for 72 h, then take 1 ml of the bacterial solution.
[0034] 2. Product extraction Freeze-dry 1 ml of the bacterial solution using a vacuum freeze dryer for 72 h until the water basically disappears, add 200 μl of methanol, vortex for 1 min using a vortex mixer, let it stand for 1 min, and repeat vortexing 5 times. Ultrasonic for 15 min, centrifuge at 10000 rpm / min for 10 min, and take the supernatant. Filter it using a 0.22-μm nylon filter membrane to prepare the sample.
[0035] 3. Product identification Identification instrument: Agilent 8890-7010B GCMSMS gas chromatography tandem triple quadrupole mass spectrometry. Chromatographic column: DB-HeavyWax (30.0m * 0.25mm * 0.25μm) capillary column. Temperature programming: The initial temperature is 50°C and is held for 2 min; it is heated to 320°C at a rate of 15°C / min and held for 5 min. Electron impact source: 70 eV, EI ion source temperature: 280°C. Injector temperature: 250°C, injection volume: 1.0 μL, injection mode: 10:1 split injection. Agilent Qualitative Navigator B.08.00 is used for analysis and quantification. The results show that the content of benzohydroxamic acid in 2×YT medium reaches about 0.15 mg / L, which is higher than 0.05 mg / L in TSB medium.
[0036] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.
Claims
1. Use of a benzohydroxamic acid biosynthetic strain in the production of benzohydroxamic acid, characterized in that: Its accession number is CGMCC NO: 4.1918.
2. A method for preparing benzohydroxamic acid, characterized in that: The steps include: S1: culturing a strain with a preservation number of CGMCC NO: 4.1918 to produce benzohydroxamic acid; optionally, further comprising: S2 extracts the produced benzohydroxamic acid.
3. The method according to claim 2, characterized in that Step S1 specifically comprises: fermenting and culturing the strain in TSB medium or 2×YT medium to produce benzohydroxamic acid; The TSB medium formula is: tryptone 17g, soy peptone 3g, NaCl 5g, glucose 2.5g, K2HPO4 2.5g, water 1000ml; The formula of the 2×YT medium is 16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride, and 1000 ml of water.
4. The method according to claim 3, characterized in that Step S1 is as follows: the strain is cultured in TSB medium at 25-35°C and 160-300 rpm / min shaking until small particles are formed; the bacterial liquid is added to TSB medium or 2×YT medium, and cultured at 25-35°C and 160-300 rpm / min shaking until small particles are formed.
5. The method according to claim 2, characterized in that Before step S1, a single clone of the strain was picked from Gao's No. 1 plate, and after verification by 16sRNA, it was added to TSB medium for culture; The formula of Gao's medium No. 1 is: 20g starch, 0.5g NaCl, 1g KNO3, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 0.01g FeSO4·7H2O, 15g Agar, and 1000ml water.
6. The method according to claim 2, characterized in that The product extraction method described in step S2 is: freeze-dry the obtained bacterial solution using a vacuum freeze dryer until the water content is basically eliminated, add methanol and vortex with a vortexer, let it stand, sonicate, centrifuge, and take the supernatant.
7. The method according to claim 4, characterized in that Repeat vortexing 5 times, ultrasonication for 12-18 minutes, centrifugation at 8000-12000 rpm / min for 8-12 minutes, and take the supernatant.
8. The method according to any one of claims 4 to 7, characterized in that The method further includes filtering with a 0.22 um nylon filter membrane and preparing samples.