Preparation method and use of a chaetomium globosum metabolite

Abstract

The present application belongs to the field of biotechnology, and particularly relates to a preparation method and application of five compounds (1-5) extracted from liquid fermentation of Chaetomium sp. Specifically, the present application discloses five compounds, wherein compound 1 is a new compound. The Chaetomium sp. metabolite has good antibacterial activity on three tested pathogenic bacteria (Pseudomonas syringae pv. actinidiae, Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola), and has important significance for development of a new type of microbial source antibacterial agent.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the preparation method and antibacterial activity study of five monomeric compounds extracted from the liquid fermentation product of Chaetomium sp. Background Technology

[0002] Plant pathogenic bacteria are second only to fungi in their harmfulness, becoming a significant threat to agricultural production and causing severe crop yield reductions and enormous economic losses globally every year. These pathogens are not only widely spread and highly destructive, but also extremely difficult to control. Faced with the dual challenges of increased resistance to chemical fungicides and rising environmental pollution and costs, microbial pesticides, with their unique advantages of being green, safe, and less prone to resistance, have become a strategic consensus for replacing chemical pesticides and ensuring agricultural product and ecological safety.

[0003] Fungal secondary metabolites are an important source of natural antibacterial active substances, possessing novel structures and diverse mechanisms of action, opening up new avenues for solving the challenges of controlling plant pathogenic bacteria. *Chaetoceros* fungi, as a widely distributed and ecologically adaptable filamentous fungus, produce secondary metabolites with rich structural types and outstanding biological activity, demonstrating great application potential in antibacterial and plant disease control fields. The abundant secondary metabolites produced by this fungus provide an important foundation for the development of highly efficient, low-toxicity novel antibacterial lead compounds. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a metabolite of Chaetomium sp. and its use.

[0005] The *Chaetoceros* strain described in this invention is specifically the strain with accession number CCTCCAF 2015035, preserved at the China Center for Type Culture Collection.

[0006] The present invention also provides a method for preparing the fermentation broth of Chaetomium sp., comprising the following steps: 1) Activated Chaetomium sp. was inoculated into PD liquid medium and cultured at 28℃ and 180 rpm for 7 days as seed culture.

[0007] Note: Activation refers to activation on PD solid medium; specifically, the bacterial cells are taken from the PD slant of the preserved strain and inoculated onto fresh PD solid medium, then incubated upside down in a 28°C incubator for 3-5 days to obtain activated Chaetomium sp. Generally, inoculating an activated bacterial block (approximately 2-3 g) into 100 mL of PD liquid medium and culturing under the above conditions will yield the seed culture.

[0008] 2) Inoculate the seed culture into PD liquid medium at a volume ratio of 1% - 2% and culture at 28℃ and 180rpm for 7 days to obtain the fermentation broth.

[0009] The present invention also provides a method for preparing metabolites using the above-mentioned Chaetomium sp. fermentation broth, comprising the following steps: 1) Filter the fermentation broth of Chaetomium sp. through 4 layers of gauze to obtain the filtrate; extract the filtrate with ethyl acetate, concentrate and dry under vacuum (at a vacuum degree of 0.1 negative pressure, 45℃) to obtain the crude extract of Chaetomium sp. fermentation broth; 2) The crude extract of Chaetomium sp. fermentation broth was crudely separated by silica gel column chromatography, using a gradient elution of dichloromethane / methanol, wherein the volume ratio of dichloromethane to methanol was 100:0, 100:1, 100:2, 100:4, 100:8, 100:16, and 100:32.

[0010] This invention also provides five Chaetomium sp. metabolites prepared using the above method, each having the following structural formula:

[0011] This invention also provides the use of the above-mentioned Chaetomium sp. metabolites: Compound 1, at a concentration of 30 μg / 6 mm filter paper, exhibited weak inhibitory activity against *Pseudomonas syringae* pv. actinidiae (Psa) and *Xanthomonas oryzae* pv. oryzae (Xoo) of rice bacterial leaf blight, with inhibition zone diameters (IZD) of 8.4 and 7.4 mm, respectively. Compound 3 exhibited strong inhibitory activity against *Psa* and *Xanthomonas oryzae* pv. oryzicola (Xoc) of rice bacterial leaf streak, with IZDs of 14.2 and 15.8 mm, respectively, slightly weaker than the positive control gentamicin (IZD = 27.5 and 20.7 mm). Compound 4 exhibited moderate inhibitory activity against Psa, Xoo, and Xoc, with IZD values ​​of 14.7, 14.2, and 16.4 mm, respectively, slightly lower than the positive control gentamicin (IZD = 27.5, 27.0, and 20.7 mm). Compounds 2 and 5 both showed weak inhibitory activity against Xoc.

[0012] Specifically, the first objective of this invention is to provide a culture of the aforementioned Chaetomium sp. strain; the second objective of this invention is to provide a method for preparing the aforementioned culture; the third objective of this invention is to provide the use of the aforementioned culture; and the fourth objective of this invention is to provide a microbial fungicide that inhibits the growth of plant pathogenic bacteria.

[0013] In summary, this invention provides a method for isolating and identifying the metabolites of Chaetomium sp., which exhibit varying degrees of inhibitory activity against bacterial canker of kiwifruit (Psa), bacterial blight of rice (Xoo), and bacterial leaf streak of rice (Xoc), laying an important foundation for the development of novel microbial fungicides. Specific implementation methods

[0014] The invention will be further explained below with reference to specific implementation examples.

[0015] Example 1: Liquid fermentation of Chaetomium sp.: Activation of *Chaetomium sp.*: Cells were taken from the PD slant culture of the preserved strain and inoculated onto fresh PD solid medium. The culture was incubated upside down at 28°C for 3-5 days to obtain activated *Chaetomium sp.*. Approximately 2-3 g of the activated strain was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of PD liquid medium and cultured at 28°C and 180 rpm for 7 days to obtain the seed culture. Then, at a 1% (v / v) inoculation rate, 5 mL of the seed culture was transferred into a 1000 mL Erlenmeyer flask containing 400 mL of PD liquid medium and cultured at 28°C and 180 rpm for 7 days to obtain the fermentation broth.

[0016] Example 2: Isolation and purification of Chaetomium sp. metabolites: 16 L of fermentation broth prepared according to the method described in Example 1 was filtered through four layers of gauze to obtain a filtrate. The filtrate was extracted three times with an equal volume of ethyl acetate. The extract was then concentrated and dried under vacuum (at a vacuum of 0.1 negative pressure and 45°C) to obtain a crude extract. The crude extract was separated by silica gel column chromatography (200-300 mesh silica gel) using dichloromethane / methanol volume ratios of 100:0, 100:1, 100:2, 100:4, 100:8, 100:16, and 100:32. The elutions from different gradients were collected separately. Finally, the structures of the five compounds were identified using a combination of spectroscopic techniques.

[0017] The spectral data of the above five compounds are as follows: Compound 1: White powder; High-resolution mass spectrometry analysis showed that its quasi-molecular ion peak m / z was 275.1263 [M+Na]+, consistent with the theoretically calculated value of 275.1260 for the molecular formula C14H20O4+; 1H NMR (600 MHz, Methanol-d4) δ: 6.04 (s, 1H, H-3), 5.53 (dd, J = 15.4, 7.6 Hz, 1H, H-9), 5.43 (dd, J = 15.5, 6.7 Hz, 1H, H-10), 4.89 (s, 4H, H-8), 4.38 (q, J = 6.8 Hz, 1H, OH-8), 2.66 (dd, J = 14.2, 7.6 Hz, 1H, H-7), 2.59 (dd, J = 14.3, 6.0 Hz, 1H, H-7), 2.02 (p, J = 6.9 Hz, 1H, H-11), 1.85 (s, 3H, H-15), 1.36 – 1.24 (m, 2H, H-12), 0.94 (d, J = 6.7 Hz, 3H, H-14), 0.85 (t, J = 7.4 Hz, 3H, H-13). 13C NMR (101 MHz, Methanol-d4) δ 167.65 (C-2), 166.27 (C-6), 160.09 (C-4), 137.64 (C-10), 129.82 (C-9), 101.65 (C-3), 97.85 (C-1), 69.86 (C-8), 41.35 (C-7), 37.85(C-11), 29.23 (C-12), 18.99 (C-14), 10.60 (C-13), 6.84 (C-15). Compound 2: White solid; high-resolution mass spectrometry showed a quasi-molecular ion peak m / z value of 227.1295 [M+H]+, consistent with the theoretical value of 227.1283 for the molecular formula C12H18O4+; ¹H NMR (600 MHz, Acetone-d6) δ: 4.68 – 4.57 (m, 1H, H-2), 3.78 (dd, J = 7.6, 2.9 Hz, 1H, H-4), 3.60 (q, J = 7.1 Hz, 3H), 2.79 (dqd, J = 13.4, 6.7, 4.4 Hz, 1H, H-6), 2.54 (ddd, J = 14.5, 7.6, 5.5 Hz, 1H, H-3β), 2.25 (t, J = 13.4). Hz, 1H, H-5β), 1.77 (dd, J = 13.6,4.5 Hz, 1H, H-5α), 1.64 (s, 3H, H-10), 1.60 (s, 1H, H-3β), 1.37 (d, J = 6.4Hz, 3H, H-9), 1.17 (t, J = 7.1 Hz, 5H), 1.11 (d, J = 6.8 Hz, 3H, H-11).13CNMR (151 MHz, Methanol-d4) δ: 203.65 (C-7), 170.16 (C-8a), 115.24 (C-8),71.12 (C-2), 70.66 (C-4), 70.52(C-4a), 40.96 (C-5), 39.01(C-3), 36.29 (C-6), 22.65 (C-9), 15.74 (C-11), 7.98(C-10).

[0018] Compound 3: A yellow oily substance; high-resolution mass spectrometry analysis showed its quasi-molecular ion peak m / z was 415.1317 [M+H]+, consistent with the theoretically calculated value of 415.1312 for the molecular formula C23H24ClO5+; 1H NMR (600 MHz, CDCl3) δ: 8.04 (s, 1H, H-1), 6.55 (m, 1H, H-20), 6.53 (s, 1H, H-4), 6.06 (d, J = 15.6Hz, 1H, H-9), 2.28 (dq, J = 13.9, 6.9 Hz, 1H, H-11), 1.90 (d, J = 6.9 Hz, 1H, H-14), 1.87 (s, 1H, H-22), 1.71 (s, 1H, H-15), 1.43 (dq, J = 12.5, 7.0 Hz,1H, H-12), 1.08 (d, J = 6.7 Hz, 1H, H-14), 0.89 (t, J = 7.4 Hz,1H, H-13);13CNMR(150 MHz, CDCl3) δ: 190.24 (C-18), 183.63 (C-6), 167.33 (C-16), 159.10 (C-8), 157.22 (C-3), 148.27 (C-1), 147.82 (C-10), 146.62 (C-20), 139.26 (C-4a),137.67 (C-19), 126.16 (C-17), 119.80 (C-9), 110.77 (C-8a), 109.54 (C-5), 105.09 (C-4), 87.56 (C-7), 38.91 (C-11), 29.10 (C-12), 25.54 (C-15), 19.25(C-14), 15.36(C-21), 11.61(C-13), 10.75(C-22).

[0019] Compound 4: Orange-red crystals; high-resolution mass spectrometry showed its quasi-molecular ion peak m / z value was 433.1408 [M+ H]+, consistent with the theoretical value of 433.1418 for the molecular formula C23H26ClO6+; 1H NMR (600 MHz, CDCl3) δ: 8.78 (s, 1H, H-1), 6.61 (dd, J = 15.5, 7.9 Hz, 1H, H-10), 6.55 (s, 1H, H-4), 6.08 (dd, J = 15.6 Hz, 1H, H-9), 3.86 (m, 1H, H-5′), 3.64 (m, 1H, H-4′), 2.29 (m, 1H, OH-5′), 1.70 (s, 3H, Me-7), 1.45 (m, 1H, H-12), 1.17 (m, 3H, Me-4′), 1.10 (d, J = 6.6 Hz, 3H, Me-11), 0.91 (m, 3H,H-13);13C NMR (150 MHz, CDCl3) δ201.05 (C-3′), 183.32 (C-6), 167.89 (C-1′), 162.82 (C-8), 157.02 (C-3), 151.49 (C-1), 148.01 (C-10), 139.57 (C-4a), 124.93 (C-2′), 119.72 (C-9),110.42 (C-8a), 109.01 (C-5), 105.31 (C-4), 87.57 (C-7), 70.90 (C-5′), 50.92(C-4′), 38.93 (C-11), 29.10 (C-12), 26.22 (7-Me), 21.47 (4′-Me), 19.24 (11-Me), 13.47 (C-6′), 11.61 (C-13).

[0020] Compound 5: A yellow oily substance; high-resolution mass spectrometry analysis showed its quasi-molecular ion peak m / z was 457.1398 [M+Na]+, consistent with the theoretically calculated value of 457.1394 for the molecular formula C23H27ClO6+; 1H NMR (600 MHz, CDCl3) δ: 7.31 (s, 1H, H-1), 6.56 (s, 1H, H-4), 6.53 (dd, J = 15.7, 8.1 Hz, 1H, H-12), 6.06 (d, J = 15.7 Hz, 1H, H-9), 4.29 (dt, J = 12.7, 6.4 Hz, 2H, H-4′), 3.04 (d, J = 9.9 Hz, 1H, H-1′), 2.99 (s, 1H, H-8), 2.25 (dt, J = 13.7, 6.8 Hz, 2H,H-11), 1.89 (dd, J = 9.8, 7.0 Hz, 1H, H-3′), 1.43 (d, J = 7.6 Hz, 1H, H-12), 1.41 (s, 3H, H-5′), 1.38 (s, 3H, Me-7), 1.11 (d, J = 6.9 Hz, 3H, Me-3′), 1.07(d, J = 6.7 Hz, 3H, Me-11), 0.90 (t, J = 7.4 Hz, 3H, H-13); 13C NMR (150 MHz, CDCl3)δ: 189.26 (C-6), 170.65 (C-1'), 157.77 (C-3), 146.87 (C-10), 145.62 (C-1), 140.55 (C-4a), 120.15 (C-9), 114.41 (C-8a), 104.96 (C-4), 104.07 (C-2'),83.81 (C-7), 58.41 (C-1'), 50.49 (C-8), 44.95 (C-3'), 38.85 (C-11), 29.15 (C-12), 23.34 (Me-7), 19.35 (Me-11), 18.68 (C-5'), 11.62 (C-13), 8.74 (Me-3').

[0021] The structural formulas of the above 5 compounds are as follows:

[0022] Example 3: Inhibitory effect of Chaetomium sp. metabolites on plant pathogenic bacteria: The antibacterial activities of compounds 1-5 against *Actinidia kiwifruit* bacterial canker (Psa), *Xoo* bacterial blight of rice (Xoo), and *Xoc* bacterial leaf streak of rice (Xoc) were detected using the filter paper disc diffusion method. The test compounds and the positive control gentamicin sulfate were prepared into 6 mg / mL solutions and sterilized by filtration through a 0.22 µm organic phase microporous membrane. The test bacteria were inoculated into their respective culture media. 200 μL of the bacterial solution was mixed with the corresponding solid culture medium, and then the medium was poured into 9 cm Petri dishes. After the medium solidified, a sterile filter paper disc (6 mm in diameter) containing 30 μg of the drug solution was placed on the surface of the solid culture medium. The medium was then inverted in a constant temperature incubator. After the test bacteria grew, the size of the inhibition zone was measured and recorded using the cross-cross method. Each experiment was repeated three times.

[0023] Table 1. Inhibition zone diameters (IZD, mm) of compounds 1-5 against tested bacteria

[0024] Notes: a: NI indicates no inhibition; b: positive control; 30 μg / 6 mm filter paper is the test concentration. Psa: Pseudomonas syringae pv. actinidae; Xoo: Xanthomonas oryzae pv. oryzae; Xoc: Xanthomnas oryzae pv. oryzicola.

[0025] The experimental results of Example 3 show that the metabolites of Chaetomium sp. have good inhibitory activity against plant pathogenic bacteria and have important potential for the development of novel microbial fungicides.

Claims

1. A method for preparing five metabolites from a strain of Chaetomium sp., characterized in that, Its preparation method includes the following steps: 1) Inoculate Chaetomium sp. into PD medium and culture at 28℃ and 180 rpm for 7 days to obtain seed culture; 2) Inoculate the seed culture into PD medium and incubate at 28℃ and 180 rpm for 7 days to obtain the fermentation broth; 3) Filter the fermentation broth obtained in step 2), extract the filtrate with ethyl acetate, concentrate and dry under vacuum to obtain crude extract; 4) Using the coarse leaching from step 3), perform silica gel column chromatography fractionation, and use dichloromethane / methanol gradient elution, wherein the volume ratio of dichloromethane to methanol is 100:1, 100:2, 100:4, 100:8, 100:16, and 100:32 to obtain different eluted fractions. 5) The obtained fraction was repeatedly passed through silica gel column, gel column, etc. to further purify the monomeric compounds, and finally five monomeric compounds were obtained.

2. The method for preparing the compound according to claim 1, characterized in that, The fermentation medium consisted of the following components per L: 200 g peeled potatoes (cut into small pieces, boiled in water for 30 min and then filtered through gauze), 20 g glucose, and water to a final volume of 1 L, pH 7.

2.

3. The use of the compound according to claim 1 in the preparation of plant pathogenic bacteria, characterized in that: wherein the new Compound 1, at a concentration of 30 μg / 6 mm filter paper, showed weak inhibitory activity against *Pseudomonas syringae* pv. *actinidiae* (Psa) and *Xanthomonas oryzae* pv. *oryzae* (Xoo), the causal agent of bacterial canker in kiwifruit, with inhibition zone diameters (IZD) of 8.4 and 7.4 mm, respectively. Compound 3 showed strong inhibitory activity against *Psa* and *Xanthomonas oryzae* pv. *oryzicola* (Xoc), the causal agent of bacterial leaf streak in rice, with IZDs of 14.2 and 15.8 mm, respectively, slightly weaker than the positive control gentamicin (IZD = 27.5 and 20.7 mm). Compound 4 exhibited moderate inhibitory activity against Psa, Xoo, and Xoc, with IZD values ​​of 14.7, 14.2, and 16.4 mm, respectively, slightly lower than the positive control gentamicin (IZD = 27.5, 27.0, and 20.7 mm). Compounds 2 and 5 both showed weak inhibitory activity against Xoc.

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