Amycolatopsis sp., cultures, metabolites, inoculants, and uses

Abstract

This invention discloses *Amycolatopsis* sp., its culture, metabolites, bacterial agents, and applications. The *Amycolatopsis* sp. provided by this invention has strain number TRM77291 and accession number CCTCC M 2024327 at the China Center for Type Culture Collection. This strain exhibits antibacterial activity against Erwinia, Salmonella, *Escherichia coli*, and *Pseudomonas aeruginosa*. This invention is of significant importance for the control of Gram-negative bacterial infections and the research of novel actinomycete-derived drugs.

Description

Technical Field

[0001] This invention relates to the field of microbiology. Specifically, it relates to pseudomycolic acid bacteria, cultures, metabolites, inoculants, and applications. Background Technology

[0002] Microorganisms produce a wide variety of secondary metabolites, which are an important source of natural medicines. Among the many microorganisms with strong metabolic capabilities, actinomycetes hold an irreplaceable position, especially Streptomyces. However, with the extensive screening of Streptomyces and the repeated discovery of numerous bioactive substances, the frequency of discovering novel bioactive natural products from Streptomyces secondary metabolites has become increasingly low. Research on the secondary metabolites of rare actinomycetes is undoubtedly one of the effective methods to address this challenge.

[0003] *Amycolatopsis* is a group of Gram-positive actinomycetes with high Gram+C content. It is a relatively rare group of actinomycetes, abundant in number and rich in secondary metabolites. This genus is closely associated with the discovery of antibiotics, having been considered one of the most effective producers of secondary metabolites with antibacterial, antifungal, or antiviral properties, and remains a focus of research for new drugs. Vancomycin, a relatively safe and effective antibiotic for treating severe infections caused by Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant coagulase-negative staphylococci, and enterococci, was discovered in *Amycolatopsis orientalis* in the 1950s and is still widely used in clinical treatment. Besides antibiotic production, *Amycolatopsis* also plays an important role in industry and ecology, such as in bioremediation (heavy metal immobilization, herbicide and polymer biodegradation) and biotransformation (vanillin production). Therefore, the study of *Amycolatopsis* secondary metabolites is of great significance.

[0004] From 1990 to 2020, researchers isolated 159 secondary metabolites from the genus *Amycolatopsis*, of which 45 compounds exhibited biological activity. Most of these compounds showed inhibitory effects on the growth of Gram-positive bacteria, and many were found to be active against various multidrug-resistant strains. The thiazomycin, a cyclic tetrapeptide containing a thiazole group, is a natural product isolated from *Amycolatopsis fastidiosa* and showed good antibacterial activity against tested Gram-positive bacteria. Compared to *Streptomyces*, rare actinomycetes possess the ability to synthesize novel natural products, and *Amycolatopsis* strains have shown great potential; 55 new compounds were isolated from *Amycolatopsis* alone between 2006 and 2018. In 2017, four novel peptide natural products were isolated from strain *Amycolatopsis* sp. M39 (termite-derived). The variety, quantity, and efficiency of novel active compounds in actinomycetes in recent years demonstrate that the secondary metabolic potential and research value of pseudo-amycetes should not be underestimated. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a pseudo-amycete, culture, metabolite, bacterial agent and application, and to extract chemical substances with antibacterial activity against Gram-negative bacteria from the fermentation products of the new pseudo-amycete species for use in inhibiting Gram-negative bacterial infection, or to develop drugs for inhibiting Gram-negative bacterial infection.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] The pseudoamycotic acid bacterium is Amycolatopsis sp., strain number TRM77291, and its accession number at the China Center for Type Culture Collection is CCTCC M 2024327.

[0008] The culture of pseudoamycete is the culture obtained by culturing the above-mentioned pseudoamycete TRM77291 in I SP series medium, Gao's No. 1 medium or Czapek's medium.

[0009] When culturing the above-mentioned culture of *Amylopectinobacterium thomsonii* TRM77291 in ISP series medium, Gao's No. 1 medium or Czapek's medium, the culture temperature is 4-40℃, the pH is 7-8, and the concentration of sodium chloride in the medium is 0wt%-5wt%.

[0010] The metabolites of *Amylopectinobacterium* are the metabolites of *Amylopectinobacterium* TRM77291 mentioned above.

[0011] An inoculum containing the above-mentioned pseudoamycete TRM77291, the above-mentioned culture, and / or the above-mentioned metabolites.

[0012] The aforementioned bacterial agents are those that inhibit Erwinia amyloliquefaciens, Salmonella, Escherichia coli, and / or Pseudomonas aeruginosa.

[0013] The above-mentioned bacterial agent contains Escherichia coli ATCC25922.

[0014] The application of pseudo-amylopectin bacteria refers to the above-mentioned pseudo-amylopectin TRM77291, or the above-mentioned culture, or the above-mentioned metabolites, or the above-mentioned bacterial agents, or the above-mentioned bacterial agents in any of the following applications:

[0015] (1) Inhibits Erwinia amyloliquefaciens, Salmonella, Escherichia coli and / or Pseudomonas aeruginosa;

[0016] (2) Prepare products for inhibiting Erwinia amyloliquefaciens, Salmonella, Escherichia coli and / or Pseudomonas aeruginosa;

[0017] (3) Prepare drugs for treating infections caused by Erwinia amylase, Salmonella, Escherichia coli and / or Pseudomonas aeruginosa;

[0018] (4) Prepare drugs for the prevention of infection with Erwinia amyloliquefaciens, Salmonella, Escherichia coli and / or Pseudomonas aeruginosa.

[0019] (5) Anti-Gram-negative bacteria;

[0020] (6) Preparation of products with anti-Gram-negative bacterial activity;

[0021] (7) To prepare medicines for the treatment and / or prevention of diseases caused by Gram-negative bacterial infections;

[0022] (5) Prepare iron chelate A and / or iron chelate E.

[0023] In the above applications, when preparing iron chelate A and / or iron chelate E using *Amylopectinobacterium* TRM77291, aerobic fermentation is employed; during aerobic fermentation, the aeration rate is 0.4–0.6 m³ / s per 70 L of fermentation broth. 3 The fermentation temperature was 30–37℃, and the fermentation time was 7–15 days. The pH of the fermentation substrate was 7–8, and the sodium chloride content in the fermentation substrate was 0.1 wt%–1 wt%.

[0024] In the above applications, the aeration rate for each 70L of fermentation broth during aerobic fermentation is 0.5m³. 3 The fermentation temperature was 30℃, and the fermentation time was 7 days. The pH of the fermentation substrate was 7, and the mass fraction of sodium chloride in the fermentation substrate was 0.3 wt%.

[0025] The technical solution of the present invention achieves the following beneficial technical effects:

[0026] 1. The pseudomycolic acid bacterium TRM77291 provided by this invention has abundant metabolic potential, especially in PKS (polyketide compounds), NRPS (non-ribosomal peptides), and RiPPs (ribosomal peptide compounds). After large-scale fermentation, it was found that the secondary metabolites of this bacterium have good inhibitory effects on four Gram-negative bacteria: Erwinia amyloliquefaciens, Salmonella, Escherichia coli, and Pseudomonas aeruginosa. Two siderocarrier compounds, siderochelin A and siderochelin E, were isolated by modern isolation and identification techniques. Siderochelin A is active against Escherichia coli and has a minimum inhibitory concentration of 64 μg / mL.

[0027] 2. The secondary metabolites of the pseudomycolic acid bacterium TRM77291 provided in this invention contain a number of active substances, which can provide a basis for the subsequent discovery of antibacterial drugs for Gram-negative bacteria, thereby enriching the types of drugs derived from rare actinomycetes.

[0028] Preservation Instructions

[0029] Classification and naming: Amycolatopsis sp.

[0030] Biomaterial from which ginseng is derived: TRM77291

[0031] Preservation Institution: China Center for Type Culture Collection

[0032] Abbreviation for depository institution: CCTCC

[0033] Address: Wuhan University, Wuhan, China

[0034] Deposit date: February 26, 2024

[0035] Collection Center Registration Number: CCTCC NO: M 2024327 Attached Figure Description

[0036] Figure 1 Phylogenetic tree of 16S rRNA gene of strain TRM77291 and its similar strains (NJ method) in the embodiments of the present invention;

[0037] Figure 2 The growth of strain TRM77291 at different temperatures in the embodiments of the present invention;

[0038] Figure 3 Physiological and biochemical characteristics test results of strain TRM77291 in the embodiments of the present invention;

[0039] Figure 4The polar lipid biphasic TLC staining results of strain TRM77291 in the embodiments of this invention;

[0040] Figure 5A HR Q-TOF MS mass spectrum of monomer compound 77291-50-A isolated from fermentation product of strain TRM77291 in this embodiment of the invention;

[0041] Figure 5B Structural diagram of monomeric compound 77291-50-A isolated from the fermentation product of strain TRM77291 in this embodiment of the invention;

[0042] Figure 5C HPLC chromatogram of si deroche lin A isolated from the fermentation product of strain TRM77291 in this embodiment of the invention;

[0043] Figure 5D The structural diagram of siderochelin E isolated from the fermentation product of strain TRM77291 in this embodiment of the invention;

[0044] Figure 6A Results of the perforation resistance method experiment of different elution components of fermentation product of strain TRM77291 in this embodiment of the invention;

[0045] Figure 6B HPLC chromatograms of different eluted fractions of fermentation products from strain TRM77291 in this embodiment of the invention;

[0046] Figure 6C A graph showing the trend of inhibition zone diameter changes in the perforation antagonism experiment of different elution components of fermentation product of strain TRM77291 in this embodiment of the invention.

[0047] Figure 7A Results of antibacterial activity test of siderochelin A in the fermentation product of strain TRM77291 using filter paper diffusion method in this embodiment of the invention;

[0048] Figure 7B The results of the two-fold dilution test of the antibacterial activity of siderochelin A in the fermentation product of strain TRM77291 in this embodiment of the invention. Detailed Implementation

[0049] Test strain: TRM77291, isolated from Tarim River silt. It is a Gram-positive aerobic bacterium.

[0050] Target bacteria: Erwinia amylovora, Salmonella, Escherichia coli, and Pseudomonas aeruginosa, all of which are Gram-negative bacteria.

[0051] Reagents: Chloroform, methanol, petroleum ether, ethyl acetate, n-butanol, acetone and DMSO were all domestically produced analytical grade, and small-pore resin (model MCI GEL CHP20P 75~150μm).

[0052] Instruments: Autoclave (HIRAYAMA, Japan); PCR instrument (SensoQues, Germany); Electrophoresis apparatus (Liuyi Instrument Factory, China); Gel imaging system (BIO-RAD, USA); Incubator (Boxum, Shanghai); Clean bench (Boxum, Shanghai); Pipettes (Eppendorf, USA); Shaker (Boxum, Shanghai); Rotary evaporator (Eye la, Japan); Low-temperature circulating water multi-purpose vacuum pump (DLSB-ZL, Zhengzhou Great Wall Science & Industry Trade Co., Ltd.); Electronic balance (CP series, Ohaus Instruments Co., Ltd.); High-speed benchtop centrifuge (GI-20-II, Shanghai Anting Scientific Instrument Factory); UV-Vis spectrophotometer (TU-1810, Beijing Purkinje General Instrument Co., Ltd.); Liquid chromatograph (Water2545, USA); Shimadzu LC-20AT high-performance liquid chromatograph (Shimadzu Corporation, Japan), equipped with Labs solubility... The system includes an Ions workstation, an LC-20AT binary gradient pump, an SPD-M20A UV detector, a column oven, and an autosampler.

[0053] The culture media used and their formulations are shown in Table 1.

[0054] Table 1:

[0055]

[0056]

[0057] I. Determination of the genetic taxonomic position of strains

[0058] 1. Molecular identification

[0059] The 16S rRNA gene sequence of the target strain TRM77291 was amplified by polymerase chain reaction using universal primers 27F (5'-AGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'), and the amplified products were sequenced. The sequencing results were assembled into a whole genome sequence and uploaded to the website ANI (http: / / www.ezbiocloud.net / tools / ani) to obtain the genome size and G+C content of the target strain TRM77291.

[0060] 2. Physiological and biochemical characteristics

[0061] The ISP series culture media, Gao's No. 1 culture medium, and Czapek's culture medium listed in Table 1 were selected. Strain TRM77291 was inoculated onto these media and cultured at 28°C for 7 days. Colony growth and various indicators were observed to determine the optimal growth medium.

[0062] The test results showed that strain TRM77291 could grow on all I SP media, Gao's No. 1 medium, and Czapek's medium, producing white spores on all of them. The colonies grew densely and optimally on Czapek's medium and ISP2 medium. Growth was relatively poor on ISP6 medium, with only a small amount of aerial hyphae growing. ISP2 was ultimately determined to be the optimal growth medium for this strain and was used as the medium for subsequent physiological and biochemical assays.

[0063] ① Sodium chloride, pH and temperature tolerance tests

[0064] I SP2 media containing 0 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, and 10 wt% sodium chloride were prepared. Strain TRM77291 was inoculated onto the surface of each sodium chloride-concentrated medium, with three replicates for each sodium chloride concentration gradient. The cultures were incubated at 28°C for 7 days. Based on the bacterial cell size and sporulation behavior, the sodium chloride concentration range tolerated by strain TRM77291 was determined to be 0 wt%–5 wt%. Further experiments with narrowed concentration gradients revealed that the optimal sodium chloride concentration for strain TRM77291 was 0.3 wt%.

[0065] ISP2 medium containing 0.3 wt% sodium chloride was used, and the pH was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0, with three replicates for each pH. Strains TRM77291 were inoculated and cultured at 28℃ for 7 days. The growth of the strain was observed to determine the pH range for its growth and the optimal pH for its growth. The results showed that strain TRM77291 has a pH tolerance range of 7–8, with an optimal growth pH of 7.0.

[0066] Strain TRM77291 was inoculated onto ISP2 medium with a sodium chloride concentration of 0.3 wt% and a pH of 7. The inoculated medium was then placed in constant temperature incubators at 4℃, 10℃, 16℃, 25℃, 28℃, 32℃, 37℃, 40℃, 45℃, and 50℃ for 7–10 days, with three replicates for each temperature gradient. Colony size, presence of aerial hyphae, sporulation, and presence of soluble pigments on the medium surface were observed to determine the suitable temperature range and optimal growth temperature for strain TRM77291. Some experimental results are shown below. Figure 2 As shown, strain TRM77291 can grow in the temperature range of 4–40℃, with its optimal growth temperature being 37℃.

[0067] ② Carbon source utilization

[0068] The following tested carbon sources, each with a mass fraction of 0.5 wt%, were added to carbon-free solid basal medium (i.e., the carbon source utilization basal mediums shown in Table 1 were prepared), with the medium without added carbon source serving as a blank control. The medium was incubated at 37°C for 7–10 days. Sporulation and colony growth of strain TRM77291 on different carbon source utilization basal media were observed.

[0069] Carbon sources tested: galactose, lactose, glucose, xylose, inositol, D-cellobiose, sorbitol, L-arabinose, D-mannitol, maltose, and mannosaccharides.

[0070] ③ Lipase assay

[0071] Strain TRM77291 was inoculated onto lipase verification medium supplemented with Tween 20, 40, 60, and 80, respectively, and incubated at 28°C for 7–10 days. If lipase was positive (i.e. strain TRM77291 could produce lipase), a white halo would appear around the colony; if lipase was negative, no white halo would appear around the colony.

[0072] ④ Gelatin liquefaction verification

[0073] Strain TRM77291 was inoculated onto gelatin liquefaction verification medium and incubated at 28°C for 7–10 days. Before observation, the plates were chilled at 4°C for half an hour. If gelatin liquefaction was observed, the strain was considered a gelatin liquefaction-positive strain.

[0074] ⑤ Determination of milk coagulation and peptone formation

[0075] Strain TRM77291 was inoculated into test tubes containing milk coagulation / peptone test medium (20 wt% skim milk powder dispersed in water) and incubated at 37°C for 7–10 days. If the liquid in the test tube showed clots, it was milk coagulation; if the liquid in the test tube was translucent, it was milk peptone.

[0076] ⑥ Starch hydrolysis verification

[0077] Inoculate strain TRM77291 onto starch hydrolysis verification medium. Incubate at 28°C for 7–10 days. Add 1–2 drops of iodine solution directly around the colonies on the medium. If starch hydrolysis is achieved, a clear halo will appear around the colonies.

[0078] ⑦ Verification of cellulose decomposition

[0079] Immerse one end of a sterile filter paper strip (5cm × 0.8cm) in the cellulose decomposition verification medium shown in Table 1. After the entire filter paper strip is wetted with the medium, inoculate strain TRM77291 onto the sterile filter paper strip above the surface of the medium and incubate at 28°C for 20–30 days. If strain TRM77291 can utilize cellulose, it will be able to grow and proliferate on the filter paper strip.

[0080] ⑧ Nitrate Reduction Verification

[0081] Inoculate strain TRM77291 into nitrate reduction verification medium (liquid medium) and incubate statically for 7–10 days. Add one drop each of Griess reagent A and B to the liquid medium. If the strain is positive for nitrate reduction, the medium will turn brown, orange, or red. If no color change occurs, add 1–2 drops of diphenylamine reagent. If the medium does not turn blue after adding diphenylamine reagent, the nitrate reduction is positive; if it turns blue, the nitrate reduction is negative.

[0082] ⑨ Verification of melanin production

[0083] Inoculate strain TRM77291 onto melanin production verification medium and incubate at 28°C for 7–10 days. If the area around the colony on the medium turns black, the strain is positive for melanin production; otherwise, it is negative for melanin production.

[0084] 3. Chemical characteristics

[0085] Polar lipid analysis: Polar lipids are an important component of cell membranes, and analyzing them helps to understand the physiological characteristics and environmental adaptability of bacterial species. Polar lipids were extracted from bacterial cells, separated using silica gel plate chromatography, and then the type of polar lipid was determined by colorimetric reactions.

[0086] ① Sample preparation

[0087] Add 0.1g of the test bacterial cells (strain TRM77291, derived from the same single clone) and 15mL of methanol to a 50mL centrifuge tube. Incubate in a boiling water bath for 10min, then cool. Add 10mL of chloroform, shake vigorously, and then add 2wt% sodium chloride solution until clear stratification occurs in the centrifuge tube. Shake vigorously for 10min after stratification. Centrifuge at 8000rpm for 10min, and transfer the clear lower layer to a rotary evaporator flask. Evaporate to dryness at 37℃. Dissolve the extract at the bottom of the flask in 0.5mL of a chloroform:methanol (2:1) mixture, transfer to a 1.5mL EP tube, centrifuge at 12000rpm for 5min, discard the precipitate, and obtain the test sample. Store the test sample at -20℃.

[0088] ②Spotting

[0089] Spot the sample on the lower left corner of the silicone plate (approximately 1.5 cm from the edge), and spot three silicone plates for each sample. The silicone plates for each sample are numbered Plate 1, Plate 2, and Plate 3.

[0090] ③ Preparation of developing solution

[0091] Developing solution 1 is chloroform:methanol:water = 65:25:4 (volume ratio); developing solution 2 is chloroform:glacial acetic acid:methanol:water = 80:15:12:4 (volume ratio).

[0092] ④ Expansion layer

[0093] Place the spotted silica gel plate into developing solution 1. When the liquid level is 1 cm from the top of the silica gel plate, remove it and let it air dry. Rotate the silica gel plate 90° and place it into developing solution 2. When the liquid level is 1 cm from the top of the silica gel plate, remove it and let it air dry.

[0094] ⑤ Preparation of color developer

[0095] Ninhydrin colorimetric reagent: Dissolve 0.4 g of ninhydrin in 100 mL of n-butanol and 10 mL of water;

[0096] Anisaldehyde colorimetric reagent: Prepare anisaldehyde colorimetric reagent according to the mass ratio of 95% ethanol (volume fraction): concentrated sulfuric acid: anisaldehyde: glacial acetic acid = 18:1:1:0.2;

[0097] Molybdenum phosphate colorimetric reagent: Dissolve 10g of molybdenum phosphate in 100mL of ethanol and mix well.

[0098] ⑥ Color development

[0099] Ninhydrin, anisaldehyde, and molybdenum phosphate were sprayed onto the surfaces of plates 1, 2, and 3, respectively. The color development of the three plates was observed, and the polar lipid type was determined.

[0100] II. Isolation, purification, and identification of natural products from bacterial strains

[0101] ① Preparation of fermented crude materials

[0102] Preparation of seed solution:

[0103] Prepare 1L of Gao's No. 1 liquid culture medium as seed culture medium. Pick a single colony of the strain that has been activated from a glycerol tube in advance and inoculate it into the seed culture medium. Incubate at 30℃ and 120r / min for about 72h to obtain seed liquid.

[0104] Fermentation in fermentation tanks:

[0105] Millet culture medium was selected as the fermentation medium, and 70 L of millet culture medium was prepared and added to the fermenter. Simultaneously, 3 mol / L NaOH and HCl solutions were prepared to adjust the pH. The temperature inside the fermenter was raised to 121℃ and maintained for 30 min to sterilize the millet culture medium. After sterilization, the temperature was cooled to 30℃, and the seed culture was inoculated into the millet culture medium. Fermentation was then carried out at 30℃ for 7 days, with a 0.5 m... 3 Sterile air is introduced into the fermenter at a rate of / h, and samples are taken for observation every two days.

[0106] Preliminary treatment of fermentation products:

[0107] The 70L fermentation broth obtained after fermentation in the above fermenter was spray-dried to obtain dry powder. Methanol was added to the dry powder, and the mixture was repeatedly extracted with methanol three times. The liquids after the three extractions were combined, and the extract was concentrated and dried at 60°C using a vacuum rotary evaporator until the methanol evaporated, to obtain the crude fermentation product.

[0108] ② Isolation, purification and identification of natural products

[0109] The crude fermentation product obtained after concentration and drying was dissolved in pure water and subjected to reversed column chromatography (MCI GEL CHP 20P 75–150 μm) using a wet loading method. Elution was performed sequentially with pure water, 30% MeOH, 50% MeOH, 70% MeOH, and 100% MeOH, with each elution volume being three times the column volume. 400 mL of each eluent was collected as one fraction, and eight fractions were collected for each eluent. After elution and concentration, each fraction was dried and analyzed by HPLC. Identical fractions were combined and concentrated again by rotary evaporation. Following the principle of "like dissolves like," elution and crystallization were performed to finally obtain the monomeric compound.

[0110] use 1 H-NMR, 13 The monomeric compounds were analyzed using nuclear magnetic resonance spectroscopy such as C-NMR, with deuterated methanol as the solvent and tetramethylsilane as the internal standard. The results were compared with published microspectral data (using http: / / www.nmrdata.com / ). The relative molecular mass of the compounds was then determined using mass spectrometry, with methanol as the solvent, to further verify the structure of the obtained monomeric compounds.

[0111] III. Bioactivity Assay

[0112] The activity of different components obtained after column chromatography and elution of the fermentation crude material was determined using the perforation antagonism method. Each component eluted from the MCI column was concentrated by rotary evaporation until the liquid components were evaporated to dryness. The concentrated components were then dissolved in an equal volume of sterile water to obtain sample solutions. Suspensions of Erwinia amyloliquefaciens, Salmonella, Escherichia coli, and Pseudomonas aeruginosa, prepared in advance, were inoculated into LB solid medium at approximately 40°C at a 1% inoculation volume. After gentle shaking, the medium was poured into plates. Once the plates solidified, a well was punched in the solid medium using a sterile 1 mL pipette tip. 150 μL of sample solution was added to each well. Three parallel experiments were conducted for each treatment. The medium was incubated at 37°C, and the size of the inhibition zone was observed.

[0113] The bioactivity of the fermented crude products was preliminarily determined using the filter paper diffusion method. Monomer compounds isolated during the separation, purification, and identification of the aforementioned natural products were prepared as 1 mg / mL methanol solutions. 50 μL of each solution was added to filter paper discs of the same size, and after the discs air-dried, they were affixed to LB agar inoculated with *E. coli*. Three parallel experiments were conducted for each treatment, with a control group consisting of filter paper discs with only methanol added. The culture medium was incubated at 37°C, and the size of the inhibition zone was observed. The size of the inhibition zone was used to represent the bioactivity of the corresponding monomer compound. After the bioactivity determination, the minimum inhibitory concentration (MIC) of each monomer compound was determined using the micro-dilution method, ultimately determining the effective inhibitory concentration range and MIC for each monomer compound.

[0114] IV. Results of Polymorphic Classification Identification of Strains

[0115] 1. Molecular identification of strain TRM77291 based on 16S rRNA sequence and whole genome information.

[0116] Using primers 27F and 1492R, the 16S rRNA gene of strain TRM77219 was cloned and sequenced. The sequencing result is shown below.

[0117] TRM77291-16S rRNA sequence (SEQ ID NO.1):

[0118]

[0119] The sequencing results were uploaded to EZb i oc l oud for comparison. The results showed that TRM77219 had a 99.49% similarity to the most similar strain, Amycolatopsis speibonae, followed by Amycolatopsis lurida (99.35%) and Amycolatopsis thailandensis (99.15%). Phylogenetic analysis was performed on the 16S rRNA sequence information using MEGA X, and a phylogenetic tree of this strain was constructed (e.g., Figure 1 As shown in the figure, strain TRM77291 forms a separate branch. To further clarify the genetic taxonomic position of the strain, second-generation sequencing analysis was performed. The results showed that TRM77291 had an ANI value of 93.93% and a dDDH value of 58.50% with the second similar strain A. lurida; and an ANI value of 90.60% and a dDDH value of 42.00% with the third similar strain A. thailandensis. The ANI and dDDH values ​​of strain TRM77291 and similar strains were all within the interspecies threshold (ANI < 95%, dDDH 20%–60%). Therefore, strain TRM77291 may be a potential new species of the genus Amycetes.

[0120] 2. Determination of physiological and biochemical characteristics

[0121] Using ISP2 medium as the basal medium, by changing the sole carbon source, it was found that strain TRM77291 can utilize any one of galactose, lactose, glucose, xylose, inositol, D-cellulose, sorbitol, L-arabinose, D-mannitol, maltose, and mannosaccharides as the sole carbon source for growth. The results are shown in Table 2. Figure 3 The image shows partial test results for the physiological and biochemical characteristics of strain TRM77291. Strain TRM77291 has the ability to peptone milk, liquefy gelatin, and reduce nitrates; it can decompose Tween 20, Tween 40, Tween 60, and Tween 80; it has the ability to produce lipase; it does not have the ability to hydrolyze starch; it cannot decompose cellulose; and it does not produce hydrogen sulfide. Figure 3 The color around the colony is due to the purple pigment and melanin produced by the strain.

[0122] Table 2 Summary of TRM77291 and its physiological and biochemical indicators

[0123]

[0124]

[0125] Note: + indicates that the indicator is positive, and the number of + indicates the strength of the ability; - indicates that the indicator is negative.

[0126] 3. Determination of chemical characteristics

[0127] Analysis using ninhydrin reagent, anisaldehyde chromogenic reagent, and molybdenum phosphate chromogenic reagent revealed that the cell membrane phospholipids of strain TRM77291 consisted of phosphatidylinositol sphos (PI), phosphatidylinositol-myo-inositol marmoside (PIM), two unknown phospholipids (PL), and two unknown glycolipids (GL). Figure 4 As shown in the figure. A represents anisaldehyde color development, B represents ninhydrin color development, and C represents molybdenum phosphate color development. The figure shows that the cell membrane of strain TRM77291 contains phosphatidylinositol (PI), phosphatidylinositol mannoside (PIM), two unknown phosphate lipids (PL), and two unknown glycolipids (GL).

[0128] Based on molecular, physiological, biochemical, and chemical characteristics, strain TRM77291 was found to differ from its similar strains in certain physiological and biochemical aspects. Its optimal growth temperature (37℃) and pH tolerance range (7–8) differ from those of similar strains; the strain possesses the ability to liquefy gelatin and peptone milk. Comparison with similar strains in physiological, biochemical, and chemical characteristics revealed differences between *A. speibonae*, *A. lurida*, and *A. thailandensis* and strain TRM77291. *A. speibonae* and *A. lurida* lack the ability to liquefy gelatin, and their optimal growth temperature and pH tolerance range are lower than those of TRM77291. The main components of their cell membranes also show some differences. In conclusion, strain TRM77291 can be inferred to be a new species within the genus *Amylopectinobacter*.

[0129] V. Isolation and Identification of Natural Products from Strains

[0130] Using the method described in the "Preliminary Treatment of Fermentation Products" section above, 70 L of millet culture medium was prepared. Strain TRM77291 was inoculated into the medium and fermented. The fermentation broth was spray-dried, yielding a total of 504 g of powder. This powder was repeatedly extracted with methanol, combined, and concentrated. The methanol was then evaporated to dryness, yielding the crude fermentation product. The crude fermentation product was dissolved in pure water and subjected to reversed-column chromatography using a wet loading method. Elution was performed sequentially with pure water, 30% MeOH, 50% MeOH, 70% MeOH, and 100% MeOH. HPLC analysis was then performed, and identical components were combined and concentrated again by rotary evaporation. During rotary evaporation, crystallization was observed in the 50% MeOH component. After complete drying of this component, it was dissolved in methanol. A suitable amount of dichloromethane was added to the methanol solution of the 50% MeOH component, resulting in pale yellow crystals at the bottom of the flask, yielding the monomer compound 77291-50-A, totaling 1040 mg.

[0131] Compound 77291-50-A was dissolved in deuterated methanol and then utilized... 1 H-NMR, 13 The NMR data were analyzed using nuclear magnetic resonance spectroscopy techniques such as C-NMR and were presented in Table 3. 13 Comparison of C-data with published microspectral data showed that the compound had a 90.9% similarity to the known compound si deroche lin A (iron chelate A).

[0132] Table 3:

[0133]

[0134] The relative molecular mass of compound 77291-50-A was then determined using mass spectrometry to further verify the structure of the obtained compound. Figure 5A The image shows the HR Q-TOF MS mass spectrum of compound 77291-50-A. The HR Q-TOF MS data of the compound in the image is (m / z 236.1042 [M+H]). + 258.0862 [M+Na] + (and data reported in the literature (m / z 236.1313 [M+H])) + 258.1138[M+Na] + The results are largely consistent, confirming that the molecular formula of compound 77291-50-A is C. 11 H 13 N3O, the structure of this compound is as follows Figure 5B As shown. Compound 77291-50-A 1The 1H NMR data are consistent with those reported in the literature as si deroche lin A, indicating that compound 77291-50-A is si deroche lin A. Figure 5C This is the HPLC chromatogram of the compound Si deroche l in A extracted in this example (detection method: 0-40 min: 10-100% MeOH; 40-50 min: 100% MeOH; 50-60 min: 10% MeOH).

[0135] Furthermore, the third eluent fraction from 50% MeOH was subjected to semi-preparative separation, followed by isocratic elution with 50% MeOH-H2O, to further separate the homologue of si deroche lin A, si deroche lin E. The structure of si deroche lin E is as follows: Figure 5D As shown.

[0136] VI. Determination of the bioactivity of fermentation products

[0137] The antagonistic activity of different elution fractions of the crude fermentation product of strain TRM77291 against Erwinia amyloliquefaciens, Salmonella, Escherichia coli, and Pseudomonas aeruginosa was tested using the perforation antagonism method. The results showed that fractions 50%-2 (representing the second fraction obtained after elution with 50% MeOH, the same below), 50%-3, 50%-4, 50%-5, ​​and 50%-6 all exhibited antagonistic activity against all four pathogens. Figure 6A As shown. Figure 6B HPLC spectra of different eluting components; Figure 6C The graph shows the trend of inhibition zone diameter in the culture medium for the perforation antagonism method. In the graph, 2, 3, 4, 5, and 6 represent components 50%-2, 50%-3, 50%-4, 50%-5, ​​and 50%-6, respectively. Table 4 shows... Figure 6C The corresponding data on the diameter of the inhibition zone.

[0138] Table 4:

[0139] 50% - 2 (cm) 50% - 3 (cm) 50% - 4 (cm) 50% - 5 (cm) 50% - 6 (cm) Pseudomonas aeruginosa 2.40 2.59 2.55 1.98 1.95 E. coli 3.03 2.20 2.17 2.22 2.11 salmonella 2.46 2.43 2.23 1.87 1.94 Erwinia amyloliquefaciens 2.44 2.31 2.12 1.89 1.98

[0140] Depend on Figure 6C It can be seen that the inhibitory effect on Pseudomonas aeruginosa ( Figure 6C (Indicated by the blue line) Component 3 showed a slightly better inhibitory effect than component 2, while components 4, 5, and 6 showed a gradual decreasing trend in inhibitory effect on Escherichia coli. Figure 6C (Represented by the orange line), components 2, 3, 4, 5, and 6 show a decreasing trend in that order; regarding the inhibitory effect on Salmonella, components 2, 3, 4, 5, and 6 show a decreasing trend in that order. Figure 6C(Indicated by the bright yellow line); Inhibitory effect on Erwinia amyloliquefaciens ( Figure 6C (Represented by the green line), components 2, 3, 4, 5, and 6 show a decreasing trend in that order; combined with... Figure 6B and Figure 6C It can be seen that the size of the inhibition zone gradually decreases as the concentration of the peak at a retention time of around 17.5 min decreases.

[0141] To further investigate the bioactivity of siderochelin A in the fermentation products of strain TRM77291, this experiment used the filter paper diffusion method to test the antagonistic activity of siderochelin A in the fermentation products against Escherichia coli ATCC25922. Figure 7A As shown, filter paper soaked in 1 mg / mL sideroche lin A methanol solution (“+” in the figure) was attached to the surface of a culture medium coated with Escherichia coli, and filter paper soaked in methanol (“-” in the figure) was used as a control. It was found that sideroche lin A had antibacterial activity against Escherichia coli ATCC 25922, and the diameter of the inhibition zone was 11±3 mm. Figure 7B To determine the minimum inhibitory concentration (MIC) of siderochelin A against target bacteria using the micro-dilution method, row a shows the results after culturing for 12–16 days with different concentrations of siderochelin A added. The siderochelin A concentrations are shown in μg / mL. Row b shows the blank culture medium. In 96-well plates, it was observed that the bacterial cultures with siderochelin A solutions at concentrations of 64–256 μg / mL were clearer than those with other concentrations and the control group (without siderochelin A solution), indicating that siderochelin A can inhibit the growth of Escherichia coli at concentrations of 64–256 μg / mL, with a MIC of 64 μg / mL.

[0142] Based on the above results, the genetic classification of strain TRM77291 was clarified. The fermentation products of strain TRM77291 exhibited good antibacterial activity against four Gram-negative bacteria: Erwinia amyloliquefaciens, Salmonella, Escherichia coli, and Pseudomonas aeruginosa. Siderochelin A and siderochelin E were isolated from the fermentation products of strain TRM77291, and the antagonistic activity of siderochelin A against Escherichia coli was verified, with a minimum inhibitory concentration (MIC) of 64 μg / mL determined. HPLC analysis revealed that the antagonistic effect of secondary metabolites of strain TRM77291 against Erwinia amyloliquefaciens, Salmonella, Escherichia coli, and Pseudomonas aeruginosa gradually decreased with decreasing concentration of peaks around 17.5 min after retention time. This indicates that secondary metabolites of strain TRM77291 contain a large number of active substances. The results of this study can provide a basis for the subsequent discovery of antimicrobial drugs for Gram-negative bacteria, thereby enriching the types of drugs derived from rare actinomycetes.

[0143] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. A pseudo-amycin bacterium, characterized in that, pseudoamycin bacterium is Amycolatopsis The strain is named TRM77291 and its accession number at the China Center for Type Culture Collection is CCTCC M 2024327.

2. A culture of pseudo-amycete bacteria, characterized in that, The culture obtained by culturing the pseudo-amylopectin bacteria TRM77291 as described in claim 1 in ISP series medium, Gao's No. 1 medium or Czapek's medium; when culturing pseudo-amylopectin bacteria TRM77291 in ISP series medium, Gao's No. 1 medium or Czapek's medium, the culture temperature is 4 to 40°C, the pH is 7 to 8, and the concentration of sodium chloride in the medium is 0 wt% to 5 wt%, and the culture contains the pseudo-amylopectin bacteria TRM77291 as described in claim 1.

3. A microbial agent, characterized in that, Contains the pseudoamycete TRM77291 as described in claim 1 or the culture as described in claim 2.

4. The microbial agent according to claim 3, characterized in that, The bacterial agent is an agent that inhibits Erwinia amyloliquefaciens, Salmonella, Escherichia coli and / or Pseudomonas aeruginosa.

5. The application of pseudo-amycinobacteria, characterized in that, The application of the pseudoamycete TRM77291 of claim 1, the culture of claim 2, the inoculum of claim 3, or the inoculum of claim 4 in any of the following: (1) Inhibits Erwinia amyloliquefaciens, Salmonella, Escherichia coli and / or Pseudomonas aeruginosa; (2) Prepare products for inhibiting Erwinia amyloliquefaciens, Salmonella, Escherichia coli and / or Pseudomonas aeruginosa; (3) Prepare drugs for treating infections caused by Erwinia amyloliquefaciens, Salmonella, Escherichia coli and / or Pseudomonas aeruginosa; (4) Prepare drugs for the prevention of infection with Erwinia amyloliquefaciens, Salmonella, Escherichia coli and / or Pseudomonas aeruginosa; (5) Preparation of iron chelate A and / or iron chelate E; When preparing iron chelate A and / or iron chelate E using *Amylopectinobacterium* TRM77291, aerobic fermentation was employed; the aeration rate for each 70 L of fermentation broth was 0.4–0.6 m³ / s. 3 The fermentation temperature is 30–37℃, the fermentation time is 7–15 days, the pH of the fermentation substrate is 7–8, and the sodium chloride content in the fermentation substrate is 0.1 wt%–1 wt%.

6. The application according to claim 5, characterized in that, During aerobic fermentation, the aeration rate is 0.5 m³ / s for every 70 L of fermentation broth. 3 The fermentation temperature was 30℃, and the fermentation time was 7 days. The pH of the fermentation substrate was 7, and the mass fraction of sodium chloride in the fermentation substrate was 0.3wt%.

Images