Non-ribosomal peptide-polyketide-polyamine class bactericidal active substance Fcl-4 and application thereof

Abstract

The present application relates to a kind of brand-new microbial natural metabolite: Fcl-4, which is derived from Xenorhabdus budapestensis (Xenorhabdus budapestensis) XBD8 fermentation liquor.Purified Fcl-4 is snow white flocculent crystal, very easy to dissolve in water, its solid state is as shown in Figure 1.Fcl-4 chemical structural formula is as shown in Figure 2, chemical formula is C 65 H 119 N 15 O 12 , molecular weight is 1302.92354, and its typical secondary mass spectrum information is as shown in Figure 3.Fcl-4 is derived from microbial natural metabolite, novel structure, high biological activity, has very high potential to develop into new biological pesticide.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a natural microbial metabolite and its application. Background Technology

[0002] Microbial natural metabolites, also known as secondary metabolites, have seen increasing identification in recent years due to the rapid development of genomics, metabolomics, and bioinformatics. Some of these natural products have been proven to be effective antibiotics and are widely used, such as vancomycin and erythromycin. Symbiotic bacteria of entomopathogenic nematodes are model organisms for studying symbiotic relationships and have become a hot topic in biopharmaceutical research. These symbiotic bacteria are Gram-negative bacteria of the Enterobacteriaceae family that parasitize the intestines of entomopathogenic nematodes, including *Xenorhabdus* and *Photorhabdus*, which live in symbiosis with *Strombus stearothermiae* and *Heterobacter spp.*, respectively. Currently, various bioactive components have been isolated and identified from known pathogenic bacilli. These compounds have novel structures and high bioactivity, possessing the potential to develop novel biopesticides, such as insecticides, antifungals, antibacterial agents, and nematodes, showing great application prospects in agriculture. According to literature reports, the main bioactive compounds of pathogenic bacilli are as follows:

[0003] (1) Dithiopyrroles: These substances were isolated from nematode pathogens and have a good effect on a variety of pathogenic bacteria, especially Gram-positive bacteria. They mainly inhibit the synthesis of bacterial RNA, thereby affecting protein synthesis.

[0004] (2) Indole derivatives: These compounds have good control effects on Gram-positive and Gram-negative bacteria, and also have a significant inhibitory effect on bacterial growth. The mechanism of action is mainly through increasing the synthesis of nucleotide ppGpp, thereby inhibiting the synthesis of RNA in Gram-negative and Gram-positive bacteria.

[0005] (3) Bacteriocins: Bacteriocins have a narrow antibacterial spectrum and usually only inhibit the growth of closely related strains. Under normal conditions, their expression levels in bacteria are very low.

[0006] (4) Amide compounds: Cyclobutyramide and ethylenediamide were isolated from the nematode pathogen YL001 strain, which had a good inhibitory effect on the mycelial growth of tomato gray mold.

[0007] (5) Water-soluble benzo[a]pyrene derivatives (XCNs): These are water-soluble isocoumarin compounds and are the most important antibacterial compounds among pathogenic bacilli. They have strong inhibitory effects on Phytophthora cucumberis, Phytophthora ramieii, and Phytophthora capsici. They have broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria.

[0008] (6) Peptides. Most peptides have advantages such as strong alkalinity, thermal stability, and broad antibacterial spectrum. A family of lysine-rich cyclic lipopeptides (PAX) composed of 5 components was isolated from the F1 strain of *Bacillus nematodes*. They not only have strong inhibitory activity against pathogenic fungi in plants and humans, but also have a certain inhibitory effect on bacteria and yeast, while having no toxicity to hamster and insect cells.

[0009] One type of fabclavine is a natural product identified in the fermentation broth of the insect pathogen symbiotic bacterium Xenorhabdus spp. Studies have found that flebotrinin exhibits broad-spectrum resistance against Gram-positive and Gram-negative bacteria, pathogenic fungi, mycoplasma, nematodes, and other organisms. It has enormous development potential in the medical and agricultural fields. Currently, most research on fulva peptides is related to their biological activity and synthesis mechanism, with few studies or inventions related to industrial development. Summary of the Invention

[0010] This invention first provides a novel microbial natural metabolite compound: Fcl-4, derived from the fermentation broth of *Xenorhabdus budapestensis* XBD8. The purified Fcl-4 is a snow-white flocculent crystal, highly soluble in water, and its solid state is as follows... Figure 1 As shown. The chemical structural formula of Fcl-4 is as follows. Figure 2 As shown:

[0011] ,

[0012] The chemical formula is C 65 H 119 N 15 O 12 Its molecular weight is 1302.92354, and its typical secondary mass spectrometry information is as follows: Figure 3 As shown.

[0013] The *Xenorhabdus budapestensis* strain XBD8, also known as strain XBD101, is a symbiotic bacterium of *Strombus budapestensis* isolated from soil samples collected in Northeast China by the inventors' laboratory. Strain XBD101 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNo:22056. The function and applications of this strain are described in Chinese invention patent ZL202110766306.8, which has been granted patent rights (CN113265364B), and its full disclosure is cited in this application. Furthermore, any *XBD8* strain mentioned in this application refers to strain XBD101.

[0014] The inventors obtained a natural product, Fcl-4, from the XBD8 fermentation broth through purification and extraction. Its structure was identified as a non-ribosomal peptide-polyketide-polyamine compound. Experiments have verified that it has significant inhibitory effects on various plant pathogenic bacteria and fungi. The plant pathogenic bacteria include, but are not limited to, *Xanthomonas aeruginosa*, *Pseudomonas syringae*, and *Rhizoctonia solani*. The plant pathogenic fungi include, but are not limited to, *Rhizoctonia solani*, *Fusarium oxysporum*, *Alternaria solani*, *Sclerotinia sclerotiorum*, *Fusarium graminearum*, *Colletotrichum orbiculare*, *Fusarium pseudograminearum*, and *Rhizoctonia cerealis*. Fcl-4, derived from natural microbial metabolites, possesses a novel structure and high biological activity, making it a potential candidate for development into novel biopesticides. Experimental studies have shown that even low concentrations (e.g., 0.49 ppm) of Fcl-4 exhibit good inhibitory effects against plant pathogenic bacteria, with the inhibitory effect significantly increasing with higher concentrations. For plant pathogenic fungi, a concentration of 5 mg / L (5 ppm) of Fcl-4 also demonstrates significant inhibitory effects. Therefore, in practical applications, it is preferable to use Fcl-4 at concentrations above 5 mg / L to prepare biopesticides, such as biofungicides, achieving stable effectiveness without environmental pollution. Attached Figure Description

[0015] Figure 1 The solid state diagram of purified Fcl-4;

[0016] Figure 2 The chemical structural formula of Fcl-4 is shown.

[0017] Figure 3 This is an infographic of Fcl-4 secondary mass spectrometry.

[0018] Figure 4-1 The MIC of Fcl-4 against Xanthomonas campestris;

[0019] Figure 4-2 The MIC of Fcl-4 against radish black rot fungus;

[0020] Figure 4-3 The MIC of Fcl-4 against Pseudomonas syringae;

[0021] Figure 5 The inhibitory effect of Fcl-4 on the mycelial growth of eight plant pathogenic fungi was studied. Detailed Implementation

[0022] The present invention will be further described below with reference to embodiments, but this is not intended to limit the invention in any way. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection of the present invention.

[0023] Example 1:

[0024] Fcl-4 was derived from the fermentation broth of *Xenorhabdus budapestensis* XBD8, which is strain XBD101. XBD8 is a symbiotic bacterium of *Strombus budapestensis* isolated from soil samples collected in Northeast China by our laboratory. Strain XBD8 was independently isolated in our laboratory and exhibits highly effective antagonistic effects against various plant pathogenic fungi and bacteria.

[0025] The inventors purified and analyzed the XBD8 fermentation broth, obtaining a novel natural product compound: Fcl-4, which is a non-ribosomal peptide-polyketide-polyamine bactericidal active substance. The specific separation and purification steps are as follows:

[0026] The XBD8 strain stored in the laboratory was taken out of the -80 ℃ freezer, streaked on LB solid plates (containing 100 μg / mL Amp), and incubated in a 28 ℃ incubator for 48 h;

[0027] Single colonies were picked and inoculated into 3 mL of LB liquid medium (containing 100 μg / mL Amp) and cultured at 28 ℃ and 200 r / min for 18 h to obtain the primary seed culture.

[0028] The primary seed culture was transferred to a shake flask containing 100 mL of LB liquid medium and incubated at 28 ℃ and 200 r / min for 18 h to be used as the secondary seed culture.

[0029] The secondary seed culture was inoculated at a rate of 1% to a volume of 1000 mL / 2000 mL in shake flasks, and fermented for 48 hours.

[0030] The fermentation broth was centrifuged at low temperature (4℃) and low speed (4500rpm) to remove the bacterial cells. The supernatant was collected and adsorbed overnight with HP-20 resin. The adsorbed resin was eluted twice with 30% acetonitrile. The eluent was rotary evaporated to obtain the concentrated elution. The concentrated elution was pre-cooled at -80℃ and dried overnight using a low-temperature vacuum dryer. The dried sample was quickly dissolved and washed with 100% methanol, centrifuged to obtain the precipitate, and the precipitate was dissolved in water to obtain the pretreated sample.

[0031] Samples were purified by HPLC. Instrument: Agilent 1260; Column: Durashell C18 (L). Mobile phase: A-acetonitrile; B-0.1% trifluoroacetic acid solution (0 min-22% A, 10 min-22% A, 11 min-90% A, 17 min-90% A, 18 min-22% A, 26 min-22% A); flow rate: 1.0 ml / min; column temperature: 35 ℃; detection wavelength: UV-203 nm.

[0032] The purified Fcl-4 is a snow-white flocculent crystal, extremely soluble in water, and its solid state is as follows: Figure 1 As shown. The chemical structural formula of Fcl-4 is as follows. Figure 2 As shown, the chemical formula is C 65 H 119 N 15 O 12 The molecular weight is 1302.92354, and the secondary mass spectrometry information is as follows: Figure 3 As shown, the mass spectrometry detection conditions are as follows:

[0033] Detection instrument: LC-MS (Ion / High Performance Liquid Chromatography-High Resolution Mass Spectrometer, Q Exactive); Column: Hypersil GOLD™ VANQUISH (100 × 2.1, 1.9µm, Thermo Fisher Scientific); Method duration: 14.00 min; Runtime: 0 to 14 min; Polarity: positive; Default charge: 1; Inclusion: on; Resolution: 17500; AGC target: 2e5; Maximum IT: 100ms; Isolation window: 2.0m / z; (N)CE / stepped nce: 20, 40.

[0034] Example 2: Inhibitory effect of Fcl-4 on bacteria:

[0035] The MIC (minimum inhibitory concentration) method in the national standard YY / T0688.1-2008 was used to determine the MIC of Fcl-4 against three typical plant pathogenic bacteria: Xanthomonas spp., radish black rot fungus, and Pseudomonas syringae. The specific steps are as follows:

[0036] 1) Use an inoculation loop to dip into the bacterial suspension of Xanthomonas lanceolata, radish black rot fungus, and Pseudomonas syringae and streak it on a regular LB agar plate. Incubate at 28°C for 12-16 h.

[0037] 2) Pick single colonies of the three strains and incubate them overnight at 200 rpm / min. Then, subculture them again at a 1% inoculum.

[0038] 3) Take samples of the three strains 2-3 hours after transfer to determine their OD values. 600 Value, pending OD 600 When the OD of the diluted bacterial solution reaches 0.8-1.0, 600 Up to 0.5.

[0039] 4) Take 20 μL of OD 600 A 0.5 μL bacterial suspension was added to 180 μL of Fcl-4 (diluted twice) at different concentrations (250 ppm, 125 ppm, 62.5 ppm, 31.25 ppm, 15.63 ppm, 7.83 ppm, 3.91 ppm, 1.95 ppm, 0.98 ppm, and 0.49 ppm), with three replicates for each concentration. The mixture was thoroughly mixed and incubated at 28°C for 24 h. The OD values ​​of the three strains under different treatments were then measured using a microplate reader. 600 The minimum concentration of antimicrobial peptide that can significantly inhibit bacterial growth is MIC.

[0040] The inhibitory effects of Fcl-4 on Xanthomonas laurentii, radish black rot fungus, and Pseudomonas syringae are shown in Figure 4 (including...). Figures 4-1 to 4-3 As shown in the figure, their MICs were 0.98 mg / L, 0.49 mg / L, and 0.98 mg / L, respectively, indicating a very significant inhibitory effect.

[0041] Example 3: Inhibitory effect of Fcl-4 on fungi:

[0042] The growth rate method was used to determine the inhibitory effect of Fcl-4 on the mycelial growth of eight plant pathogenic fungi, including Rhizoctonia solani (potato black scurf), Fusarium oxysporum, Alternaria solani, Sclerotinias clerotiorum (sunflower sclerotium), Fusarium graminearum, Colletotrichumorbiculare (cucumber anthracnose), Fusarium pseudograminearum, and Rhizoctonia cerealis (wheat sheath blight).

[0043] 1) Inoculate the pathogenic fungi stored at -80℃ onto PDA plates and incubate them at 25℃ in the dark for 5 days.

[0044] 2) Add Fcl-4 to PDA medium and mix to prepare plates with concentrations of 5 ppm, 10 ppm and 20 ppm. After mixing, pour the mixture into 6 cm plates and use an equal amount of water as a blank control. Each treatment is repeated three times.

[0045] 3) The activated pathogenic fungi were inoculated onto plates containing different concentrations of Fcl-4 and cultured at 25°C in the dark for 5 days.

[0046] 4) Measure the colony diameter under each treatment using the cross-multiplication method, and calculate the mycelial growth inhibition rate of each target bacterium according to formulas (1) and (2). The unit is percentage (%), and the calculation results are retained to two decimal places.

[0047] Equation (1): D = D1 - D2

[0048] Equation (2):

[0049]

[0050] In formula (1): D: colony growth diameter; D1: colony diameter; D2: mycelium cake diameter.

[0051] In formula (2): I: mycelial growth inhibition rate; D0: colony growth diameter of blank control; Dt: colony growth diameter of different treatments.

[0052] Fcl-4 showed inhibitory effects on the mycelial growth of eight plant pathogenic fungi, including *Rhizoctonia solani* (potato black scurf), *Fusarium oxysporum*, *Alternaria solani*, *Sclerotinias clerotiorum* (sunflower sclerotium), *Fusarium graminearum*, *Colletotrichumorbiculare* (cucumber anthracnose), *Fusarium pseudograminearum*, and *Rhizoctonia cerealis* (wheat sheath blight). Figure 5 As shown in Table 1, the inhibition rates of 5 mg / L Fcl-4 against different fungi ranged from 44.3±2.4% to 97.8±1.1%, 10 mg / L Fcl-4 ranged from 63.5±5.7% to 100.0±1.1%, and 20 mg / L Fcl-4 ranged from 68.7±2.3% to 100.0±0.1%, demonstrating significant inhibitory effects.

[0053]

[0054] The above experiments show that even low concentrations (e.g., 0.49 ppm) of Fcl-4 have a good inhibitory effect on plant pathogenic bacteria, and the inhibitory effect increases significantly with increasing concentration. For plant pathogenic fungi, a concentration of 5 mg / L (i.e., 5 ppm) of Fcl-4 also has a significant inhibitory effect. Therefore, in practical applications, it is preferable to use Fcl-4 at a concentration of 5 mg / L or higher to prepare biological pesticides, such as biological fungicides, to achieve stable and effective results without causing environmental pollution.

[0055] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A non-ribosomal peptide-polyketide-polyamine bactericidal active substance Fcl-4, characterized in that, The chemical structural formula of Fcl-4 is shown below: ， The chemical formula is C 65 H 119 N 15 O 12 Its molecular weight is 1302.92354.

2. The Fcl-4 according to claim 1, characterized in that, The Fcl-4 is a snow-white flocculent crystal that is extremely soluble in water.

3. The Fcl-4 according to claim 1, characterized in that, The Fcl-4 was purified from the fermentation broth of Xenorhabdus budapestensis XBD8; the XBD8 strain is the same as the XBD101 strain, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No:22056.

4. The Fcl-4 according to any one of claims 1-3, characterized in that, The Fcl-4 has a significant inhibitory effect on plant pathogenic bacteria.

5. The Fcl-4 according to claim 4, characterized in that, The plant pathogenic bacteria mentioned are Xanthomonas aureus, radish black rot fungus, and Pseudomonas syringae.

6. The Fcl-4 according to any one of claims 1-3, characterized in that, The Fcl-4 has a significant inhibitory effect on plant pathogenic fungi.

7. The Fcl-4 according to claim 6, characterized in that, The plant pathogenic fungi mentioned are Rhizoctonia solani (potato black scurf), Fusarium oxysporum, Alternaria solani (soybean sclerotinia), Sclerotinia sclerotiorum (sunflower), Fusarium graminearum (grass spores), Colletotrichum orbiculare (cucumber anthracnose), Fusariumpseudograminearum (false grain spores), and Rhizoctonia cerealis (wheat sheath blight).

8. The use of Fcl-4 according to any one of claims 1-7 in the preparation of biopesticides, wherein the biopesticide is a biofungicide for killing plant pathogenic bacteria and plant pathogenic fungi.

9. The application according to claim 8, characterized in that, The concentration of Fcl-4 is above 5 mg / L.

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