Use of pantoea agglomerans strain zju23 in inhibiting bacteria

Abstract

The present application relates to the technical field of development and utilization of microbial germplasm resources, and particularly relates to application of Pantoea agglomerans ZJU23 strain in inhibiting bacteria. The ZJU23 strain can inhibit field bacteria of 4 phyla and 25 genera, and has broad-spectrum bacteriostasis. Then, the culture medium of the ZJU23 strain is optimized, and it is found that only when the ZJU23 strain is inoculated into the WA culture medium with glucose as a carbon source, the ZJU23 strain has the effect of inhibiting bacteria. Further, the present application proves that the ZJU23 strain achieves the effect of inhibiting bacteria by producing gluconic acid and reducing pH. In summary, the present application finds a new use of the Pantoea agglomerans ZJU23 strain, expands the application range thereof, and improves the application value thereof. In addition, the present application also explores the culture medium formula and bacteriostasis mechanism with the best bacteriostasis effect, and lays a theoretical foundation for use of the ZJU23 strain.

Description

Technical Field

[0001] This invention relates to the field of microbial germplasm resource development and utilization technology, and in particular to the application of Pantotheca acuminata strain ZJU23 in inhibiting bacteria. Background Technology

[0002] Pantotheca cumulus is widely distributed in natural ecological environments. Early studies mostly classified it as an opportunistic pathogen, a perception that significantly limited the development and utilization of this type of microbial resource. With the continuous advancement of related basic research, current studies have confirmed that Pantotheca cumulus possesses multiple excellent biological characteristics, including activation of insoluble phosphorus in soil, biological nitrogen fixation, efficient rhizosphere colonization in crops, excellent environmental stress resistance, and biological hydrogen production. In addition, Pantotheca cumulus can synthesize various functional secondary metabolites such as antimalarial alkaloids, lipopolysaccharides, and bioactive peptides. Due to these diverse application potentials, this species has now become a research hotspot in the field of microbial resources.

[0003] Chinese patent CN 110713951 B discloses a *Pantotheca acuminata* strain ZJU23 capable of simultaneously controlling diseases caused by plant pathogenic fungi such as Fusarium head blight, gray mold, rice blast, soybean anthracnose, and rice sheath blight. However, whether this strain can inhibit bacteria, the spectrum of bacterial inhibition, and the mechanism of bacterial inhibition are still unknown. Further investigation into the antibacterial function of *Pantotheca acuminata* strain ZJU23 could expand its application prospects and has significant practical implications. Summary of the Invention

[0004] To achieve the above objectives, this invention provides the application of the *Pantotheca acuminata* strain ZJU23 in inhibiting bacteria. The ZJU23 strain can inhibit field bacteria of 4 phyla and 25 genera, exhibiting broad-spectrum antibacterial activity. Furthermore, this invention optimized the culture medium for the ZJU23 strain, finding that it only exhibits antibacterial activity when inoculated onto WA medium with glucose as the carbon source. Further, this invention demonstrates that the ZJU23 strain achieves its antibacterial effect by producing gluconic acid and lowering the pH. In summary, this invention discovers a new use for the *Pantotheca acuminata* strain ZJU23, expanding its applicability and increasing its application value. In addition, this invention explores the optimal culture medium formulation and antibacterial mechanism for its optimal antibacterial effect, laying a theoretical foundation for the use of the ZJU23 strain.

[0005] The first objective of this invention is to provide a cluster of pantothenic bacteria (Pantothenic bacteria) Pantoea agglomerans The use of ZJU23 strain or its mutants, or preparations containing said ZJU23 strain or its mutants, in inhibiting bacteria or in the preparation of antibacterial products.

[0006] A second objective of this invention is to provide a method for inhibiting bacteria.

[0007] A third objective of this invention is to provide the application of Pantotheca agglutinosa strain ZJU23 or its mutants, or preparations containing said ZJU23 strain or its mutants, in the production of gluconic acid or the preparation of gluconic acid products.

[0008] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution: The first aspect of the present invention is to provide the use of Pantotheca acuminata strain ZJU23 or a mutant thereof, or a preparation containing said ZJU23 strain or a mutant thereof, in inhibiting bacteria or preparing antibacterial products. The ZJU23 strain is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 16174 and deposit date of July 30, 2018.

[0009] In some specific embodiments, the Pantotheca agglutinosa strain ZJU23 or its mutants inhibit the bacteria by any of the following means: producing gluconic acid; synthesizing pyrroloquinoline quinone (PQQ); or lowering the pH.

[0010] In some specific embodiments, the preparation containing the ZJU23 strain or its mutant further comprises a culture medium.

[0011] More specifically, the culture medium includes any one or more of WA medium, TSB medium, LB medium, LBGM medium, NA medium, M9 medium, KB medium, and R2A medium; preferably WA medium.

[0012] More specifically, the carbon source of the culture medium includes any one or more of glucose, trehalose, starch, mannose, fructose, maltose, D-arabinose, D-xylose, rhamnose, glycerol, lactose, sucrose, mannitol, dextrin, and melibiose; preferably glucose and D-arabinose; more preferably glucose.

[0013] In some specific embodiments, the bacteria are Gram-negative or Gram-positive bacteria.

[0014] In some other specific embodiments, the bacteria are selected from: Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes. More specifically, the bacteria are selected from: Ensifer, Rhizobium, Brucella, Ochrobactrum, Pseudomonas, Enterobacter, Lelliottia, Stenotrophomonas, Achromobacter, Commonas, Burkholderia, Chryseobacterium, and Rho dococcus, Kocuria, Arthrobacter, Citricoccus, Pseudarthrobacter, Streptomyces, Agromyces, Curtobacterium, Microbacterium, Paenibacillus, Priestia, Brevibacterium, Bacillus.

[0015] In some specific embodiments, the mutant of the ZJU23 strain is Δ gad1 Δ gad2 or Δ gad1 Δ gad2 Δ gnuk .

[0016] A second aspect of the invention is to provide a method for inhibiting bacteria, namely, using clumps of pantothenia (Pantothenia gravis). Pantoea agglomerans The ZJU23 strain or its mutant, or a preparation containing the ZJU23 strain or its mutant, is used to inhibit bacteria; the ZJU23 strain is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 16174 and deposit date of July 30, 2018.

[0017] In some specific embodiments, the OD of the *Pantotheca acuminata* strain ZJU23 is... 600=0.6~1.5, more preferably OD 600 =0.8~1.2, further optimization of OD 600 =1.0.

[0018] In some specific embodiments, the Pantotheca agglutinosa strain ZJU23 or its mutants inhibit the bacteria by any of the following means: producing gluconic acid; synthesizing pyrroloquinoline quinone (PQQ); or lowering the pH.

[0019] In some specific embodiments, the preparation containing the ZJU23 strain or its mutant further comprises a culture medium.

[0020] More specifically, the culture medium includes any one or more of WA medium, TSB medium, LB medium, LBGM medium, NA medium, M9 medium, KB medium, and R2A medium; preferably WA medium.

[0021] More specifically, the carbon source of the culture medium includes any one or more of glucose, trehalose, starch, mannose, fructose, maltose, D-arabinose, D-xylose, rhamnose, glycerol, lactose, sucrose, mannitol, dextrin, and melibiose; preferably glucose and D-arabinose; more preferably glucose.

[0022] In some specific embodiments, the bacteria are Gram-negative or Gram-positive bacteria.

[0023] In some other specific embodiments, the bacteria are selected from: Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes. More specifically, the bacteria are selected from: Ensifer, Rhizobium, Brucella, Ochrobactrum, Pseudomonas, Enterobacter, Lelliottia, Stenotrophomonas, Achromobacter, Commonas, Burkholderia, Chryseobacterium, and Rho dococcus, Kocuria, Arthrobacter, Citricoccus, Pseudarthrobacter, Streptomyces, Agromyces, Curtobacterium, Microbacterium, Paenibacillus, Priestia, Brevibacterium, Bacillus.

[0024] In some specific embodiments, the mutant of the ZJU23 strain is Δ gad1 Δ gad2 or Δ gad1 Δ gad2 Δ gnuk .

[0025] Glucoic acid is a naturally occurring organic acid derived from microorganisms, possessing excellent biocompatibility and complete biodegradability. In the food industry, it can be used as an acidity regulator and preservative for fruits and vegetables; in the pharmaceutical and chemical industries, it serves as an intermediate in the preparation of calcium supplements and medical antibacterial excipients; in pesticides, this substance can chelate soil metal ions, activate insoluble phosphorus in the soil, and simultaneously acidify the microenvironment and inhibit the proliferation of harmful microorganisms, making it a key active metabolite for soil improvement and green disease control through microbial agents. In view of the aforementioned beneficial effects of gluconic acid, this invention requests the use of *Pantotheca acuminata* strain ZJU23 or its mutants, or preparations containing said ZJU23 strain or its mutants, in the production of gluconic acid or the preparation of gluconic acid products.

[0026] A third aspect of the present invention is the use of Pantotheca agglutinosa strain ZJU23 or its mutant, or a formulation containing said ZJU23 strain or its mutant, in the production of gluconic acid or the preparation of gluconic acid products, characterized in that said ZJU23 strain is deposited at the China General Microbiological Culture Collection Center, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, accession number CGMCC No. 16174, deposit date July 30, 2018.

[0027] In some specific embodiments, the mutant of the ZJU23 strain is Δ gad1 Δ gad2 or Δ gad1 Δ gad2 Δ gnuk .

[0028] Compared with the prior art, the beneficial effects of the present invention include: (1) This invention provides a new use for ZJU23 strain or its mutants to inhibit bacteria and produce gluconic acid, expands the scope of application of ZJU23 strain, and improves the economic value of ZJU23 strain.

[0029] (2) This invention optimizes the culture medium of strain ZJU23, improves its antibacterial effect, and lays a theoretical foundation for its practical application.

[0030] (3) This invention elucidates the mechanism of action of ZJU23 strain in inhibiting bacteria, which not only lays a solid theoretical foundation for its practical application, but also provides new ideas for the field of bacterial prevention and control. Attached Figure Description

[0031] Figure 1 The antibacterial spectrum of Pantotheca acuminata strain ZJU23, where the scale bar = 5 mm.

[0032] Figure 2 The effect of culture medium type on the inhibitory effect of Pantotheca agglomerata strain ZJU23 on Bacillus subtilis. (Scale bar = 4 mm)

[0033] Figure 3 The effect of carbon source type on the inhibitory effect of Pantotheca agglomerata strain ZJU23 on Bacillus subtilis. (Scale bar = 4 mm)

[0034] Figure 4 The inhibitory effects of different ZJU23 strain mutants on Bacillus subtilis, among which, Figure 4 a represents the inhibitory effect of the metabolic gene cluster knockout mutant on Bacillus subtilis, scale bar = 5 mm; Figure 4b represents the inhibitory effect of the transposon insertion mutant on Bacillus subtilis, and 1~4 represent different transposon mutants. Scale bar = 5 mm. Figure 4 c is PQQ A schematic diagram of the structure of gene clusters in the ZJU23 genome.

[0035] Figure 5 :PQQ to Δ pqq The effect of the strain on the inhibition of Bacillus subtilis, where the scale bar = 5 mm.

[0036] Figure 6 pH detection of Pantotheca ZJU23 colonies, where scale bar = 4 mm.

[0037] Figure 7 Effect of PBS on the antibacterial activity of Pantotheca acuminata strain ZJU23, where the scale bar = 4 mm.

[0038] Figure 8 :PQQ to Δ pqq The effect of bacterial colony pH on the bacterial strain, where the scale bar = 5 mm.

[0039] Figure 9 The inhibitory effect of the ZJU23 mutant strain with the glucose pathway gene knocked out on Bacillus subtilis. ZJU23: wild type, as a positive control; Control: strain without any ZJU23 strain, as a negative control; scale bar = 5 mm.

[0040] Figure 10 pH detection of mutant colonies of ZJU23 strain with glucose pathway gene knocked out, where ZJU23: wild type, as positive control; Control: no ZJU23 strain, as negative control; scale bar = 5 mm.

[0041] Figure 11 The growth curves of Bacillus subtilis after the addition of strain ZJU23 or its mutant are shown. Among them, 3610 is the bacterial culture of Bacillus subtilis strain 3610 without strain ZJU23 or its mutant, which serves as a negative control; ZJU23 is the bacterial culture of Bacillus subtilis strain 3610 containing wild-type strain ZJU23, which serves as a positive control.

[0042] Figure 12 pH detection of fermentation broths of different ZJU23 strain mutants.

[0043] Figure 13 Detection of gluconic acid content in fermentation broth of different ZJU23 strain mutants.

[0044] Figure 14The effect of gluconate on inhibiting Bacillus subtilis. Detailed Implementation

[0045] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods in the art; and unless otherwise specified, three replicates are provided. Unless otherwise specified, the reagents and instruments in the following embodiments are commercially available. Unless otherwise specified, the software, parameters, and statistical methods used in the following embodiments can be adjusted according to conventional methods in the art. Modifications, substitutions, or equivalent transformations made by those skilled in the art to the present invention without departing from the essential content of the present invention should all fall within the scope of protection of the present invention.

[0046] This invention focuses on the antagonistic bacterial activity of the Pantotheca acuminata ZJU23 strain, exploring the relationship between its antibacterial phenomenon, culture conditions, carbon source metabolism, and related genes, and conducting a preliminary analysis of its possible antibacterial mechanism.

[0047] First, this invention explored the antibacterial spectrum of the Pantotheca acuminata strain ZJU23, demonstrating that it can inhibit field bacteria of 4 phyla and 25 genera to varying degrees, exhibiting broad-spectrum activity. Among these, compared with Proteobacteria, Bacteroidetes, and Firmicutes, strain ZJU23 showed better overall inhibition of Actinobacteria, especially Rhodococcus.

[0048] Next, the ability of strain ZJU23 to antagonize Bacillus subtilis under different culture medium conditions was compared. The experiment found that strain ZJU23 could significantly inhibit the growth of Bacillus subtilis 3610 on WA medium, while the inhibitory effect was weak or even disappeared on conventional media such as LB.

[0049] Furthermore, through analysis and verification of the culture medium composition, it was determined that the carbon source in the medium is a key factor affecting antibacterial activity. Therefore, the effects of various carbon sources, such as glucose, arabinose, rhamnose, and trehalose, on the activity of the strain were systematically compared. The results showed that glucose most significantly promoted the antagonistic effect of ZJU23, while arabinose also had a certain promoting effect. In further concentration optimization experiments, it was found that the antibacterial activity of the strain reached its optimum when the glucose concentration was 3.0%. In summary, this invention established a relatively stable optimized culture system, providing a reliable experimental basis for subsequent research.

[0050] In the study of antibacterial mechanisms, this invention first isolated, purified, and structurally identified the antagonistic substances of strain ZJU23, and elucidated its biosynthetic regulatory mechanism. However, during the research process, it was found that this antibacterial phenomenon was more closely related to the primary metabolism of the strain, so the focus shifted to the analysis of the antibacterial phenomenon itself and its metabolic basis. Through transposon mutant screening, four mutants with significantly reduced activity were obtained. After resequencing analysis, it was found that multiple mutation sites were concentrated in the regions of glucose metabolism and pyrroloquinoline quinone (PQQ) related genes, suggesting that the antibacterial ability of strain ZJU23 is closely related to PQQ synthesis and glucose oxidation metabolism.

[0051] In further experiments, this invention constructed Δ pqq The mutant strain was compared phenotypically with the wild-type strain. Results showed that the antibacterial ability of the strain was significantly reduced after the relevant gene was deleted. Simultaneously, observation of pH changes in the culture medium revealed that strain ZJU23 exhibited strong acid-producing ability under high glucose conditions, resulting in significant acidification of the culture environment. This acidification phenomenon was highly correlated with antibacterial ability. Therefore, it is speculated that the antagonistic effect of strain ZJU23 mainly originates from the organic acids produced during glucose metabolism and the resulting acidic environment, rather than from a single complex antibiotic-like substance.

[0052] To verify the above conjectures and identify the acidic substances secreted by strain ZJU23, this invention constructed a series of glucose metabolism pathway gene knockout mutants of strain ZJU23. It was found that only the mutant capable of producing gluconic acid (Δ...) gad1 Δ gad2、 Δ gad1 Δ gad2 Δ gnuk Only then did the acidification zone and inhibition zone appear, and Δ gad1 Δ gad2、 Δ gad1 Δ gad2 Δ gnuk The mutant fermentation broth contained a high level of gluconic acid. These experimental results fully demonstrate that strain ZJU23 inhibits bacteria by secreting gluconic acid and lowering the pH.

[0053] Example 1: Antibacterial spectrum of Pantotheca acuminata strain ZJU23 This embodiment determined the antibacterial spectrum of the Pantotheca acuminata ZJU23 strain. The specific steps are as follows: 1.1 Isolation of test bacteria Sampling location: Zhejiang University Healthy Agriculture Research Base (N 30°24′, E 120°3′36″) Take 5 g of wheat rhizosphere soil sample and add it to a centrifuge tube containing 45 mL of sterile water. Vortex for 2 min to mix thoroughly, then let stand for 5 min to form a soil suspension. Add 1 mL of the soil suspension to 9 mL of sterile water, vortex for 2 min to mix thoroughly, then let stand for 5 min to obtain 10 g of the soil suspension. -1 Dilute the soil suspension in a gradient, diluting it sequentially to obtain 10... -2 10 -3 10 -4 10 -5 Soil suspensions were diluted in a gradient. 100 μL of each dilution was spread onto LB agar (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, pH 7) using a sterile spreader. Each gradient was repeated three times. The plates were incubated upside down in a 30°C incubator for 24–48 h. After incubation, colony color and other differences were observed. Single colonies were picked and streaked for purification to obtain pure cultures. The purified strains were then preserved using the glycerol preservation method and stored at -80°C.

[0054] 1.2 Antibacterial activity test of Pantotheca acuminata strain ZJU23 Using WA medium (formulation: 5 g / L peptone, 10 g / L glucose, 3 g / L beef extract, 5 g / L sodium chloride, 15 g / L agar) as the base, OD was added to each medium. 600 =1.0 bacterial suspension obtained from field isolation (isolation steps as described above), 10 μL, was added to the midpoint of a dried plate. 600 The antibacterial activity of the *Panthera solani* strain ZJU23 (with a concentration of 1.0) was observed after incubation at 30°C for 2 days. See the attached table for details. Figure 1 Each experimental group was set up with 3 replicates.

[0055] Depend on Figure 1It can be seen that the ZJU23 strain can simultaneously inhibit 20 strains of Gram-negative bacteria and 50 strains of Gram-positive bacteria, and its antibacterial spectrum covers 4 phyla (including Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes) and 25 genera (including Ensifer, Rhizobium, Brucella, Ochrobactrum, Pseudomonas, Enterobacter, Lelliottia, Stenotrophomonas, Achromobacter, Comamonas, Burkholderia, Chryseobacterium, Rhodococcus, Kocuria, Arthrobacter, Citricoccus, Pseudarthrobacter, Streptomyces, Agromyces, Curtobacterium, Microbacterium, Paenibacillus, Priestia, Brevibacterium, Bacillus). Among them, compared with the other 3 phyla, the ZJU23 strain has a better overall effect on inhibiting bacteria of the Actinobacteria phylum, especially the genus Rhodococcus.

[0056] In general, the Pantoea agglomerans ZJU23 strain exhibits broad-spectrum activity and is suitable for the control of common field bacteria.

[0057] Example 2 Effect of medium type on the antibacterial activity of Pantoea agglomerans strain ZJU23 From Figure 1 it can be seen that the Pantoea agglomerans ZJU23 strain can inhibit a variety of bacteria of the genus Bacillus. Therefore, in this example, the model Bacillus subtilis strain 3610 (corresponding to Figure 1 the NCIB 3610 strain in the above) is used as the test bacterium to explore the effect of the medium on the antibacterial activity of the Pantoea agglomerans ZJU23 strain. The specific steps are as follows: Using different media (the specific formulations are shown in Table 1) as the base, add OD 600=1.0 Bacillus subtilis ( Bacillus subtilis 10 μL of bacterial suspension (preserved in the inventor's laboratory) was spotted in the center of a dried agar plate, and 10 μL of OD was observed. 600 The antibacterial activity of the ZJU23 strain with a bacterial concentration of 1.0 μg / mL was observed after incubation at 30°C for 2 days. Each experimental group was set up with 3 replicates. The experimental results are shown below. Figure 2 .

[0058] Table 1 Culture medium formulation

[0059] Depend on Figure 2 It is known that the Pantotheca acuminata ZJU23 strain can grow on all culture media, but only when the ZJU23 strain is inoculated onto WA medium can an inhibition zone appear around it, thus enabling it to inhibit Bacillus subtilis.

[0060] Example 3: Effect of carbon source on the antibacterial activity of Pantotheca acuminata strain ZJU23 Example 2 revealed that strain ZJU23 could only inhibit Bacillus subtilis when grown in WA medium. Based on this, analysis and verification of the medium composition determined that the carbon source in the medium was a key factor affecting the antibacterial activity. Therefore, this example uses WA medium as a basis to explore the effect of different carbon sources on the antibacterial activity of the Pantotheca agglomerata strain ZJU23. The specific steps are as follows: Using WA medium with 1% different carbon sources as the base, OD was added 600 10 μL of Bacillus subtilis 3610 bacterial suspension with a concentration of 1.0 was added to the midpoint of a dried plate. OD 600 The antibacterial activity of ZJU23 strain with a carbon content of 1.0 was observed after incubation at 30℃ for 2 days. Each experimental group was set up with 3 replicates. The carbon source sources are shown in Table 2. The experimental results are shown in [Table 2]. Figure 3 .

[0061] Table 2 Sources of carbon

[0062] Depend on Figure 3 It can be seen that strain ZJU23 can grow on WA medium with all different carbon sources, but it can only inhibit Bacillus subtilis when the carbon source is glucose or D-arabinose. Among them, the inhibitory effect is better when the carbon source is glucose. Therefore, WA medium with 1% glucose as carbon source was used for subsequent experiments.

[0063] Example 4: Investigation into the antibacterial mechanism of Pantotheca acuminata strain ZJU23 4.1 The antibacterial activity of Pantotheca acuminata strain ZJU23 is related to the synthesis of PQQ. 4.1.1 Knockout of metabolic gene clusters in Pantotheca acuminata strain ZJU23 This invention uses the λ-red gene knockout system to knock out genes in pantothenic bacteria.

[0064] (1) Acquisition of knockout fragments First, primers were designed to amplify homologous arm fragments of 500-800 bp flanking the target gene, and adapters were added to the primer junctions. Alternatively, a 50 bp homologous arm could be directly added to the 5' end of the primer. Specific primer sequences are shown in Table 3. Genomic DNA was extracted from strain ZJU23 using the bacterial genomic DNA extraction kit (DP302) from Tiangen Biotech (Beijing) Co., Ltd. Using the genomic DNA as a template, upstream and downstream homologous arms were amplified. Simultaneously, the kanamycin resistance gene was amplified using plasmid pKD4 as a template. The first-round PCR products were obtained by agarose gel electrophoresis and gel recovery. Gel recovery was performed using the SanPrep column DNA gel recovery kit (B518131) from Sangon Biotech (Shanghai) Co., Ltd. Subsequently, a second-round PCR was performed using the first-round PCR products as templates. The three fragments were fused using overlap extension PCR technology to obtain a long fragment containing the upstream homologous arm, kanamycin resistance, and downstream homologous arm. Finally, the second-round PCR products were used as templates. A third round of PCR was performed, using Taq polymerase for amplification, and the fragments were recovered by gel electrophoresis. The resulting fragments will be used in subsequent experiments.

[0065] Table 3 Primer sequences for amplifying the homologous arms of the target gene.

[0066] (2) Preparation of heat-strain competent cells of Pantotheca acuminata ZJU23 The overnight culture of ZJU23 bacteria was inoculated into 100 mL of LB liquid medium at a ratio of 1:100 and incubated at 30°C for 2–3 h until OD was reached. 600 The concentration of the bacterial culture was approximately 0.5–0.8. Subsequently, the bacterial culture was placed on ice for 30 min, centrifuged at 4000 rpm for 10 min to collect the bacterial cells, and centrifuged at 4°C. The bacterial cells were resuspended in 10 mL of pre-chilled 0.1 M CaCl2, placed on ice for 30 min again, and centrifuged at 4000 rpm at 4°C for 5 min to collect the bacterial cells. Finally, the bacterial cells were resuspended in 2–4 mL of pre-chilled 0.1 M CaCl2 containing 15% glycerol, placed on ice for a few minutes, and the competent cells were prepared. The cells were aliquoted into 100 μL tubes and stored at -80°C.

[0067] (3) pKD46 plasmid was transformed into strain ZJU23 10 μL of pKD46 plasmid was added to the prepared competent cells, incubated on ice for 30 min, then heat-shocked at 42°C for 60 s, rapidly cooled, and then 800 μL of LB medium was added. The cells were then incubated at 30°C for about 50 min. After incubation, the supernatant was collected by centrifugation and resuspended in 100 μL of LB medium. The supernatant was then plated on a plate containing 100 μg / mL ampicillin and incubated at 30°C. After colonies grew, the plasmid was verified by the corresponding primers. The correct strain ZJU23(pKD46) was preserved for the next step of the experiment.

[0068] (4) Preparation of electrocompetent cells Colonies of ZJU23(pKD46) were picked from the plate and inoculated into LB medium. The culture was incubated overnight at 30°C and 180 rpm. The overnight culture was then inoculated into 100 mL of LB medium at a 1:100 ratio, and ampicillin (to a final concentration of 100 μg / mL) and L-arabinose (to a final concentration of 20 mM) were added. The culture was incubated at 30°C and 180 rpm for approximately 3.5–4 h. After shaking, the culture was placed on ice for 30 min, centrifuged at 4000 rpm for 10 min at 4°C to collect the cells. The cells were then resuspended in pre-chilled ddH2O, placed on ice for another 30 min, and centrifuged at 4000 rpm for 6 min at 4°C to collect the cells. This process was repeated three times to remove ions from the solution. Finally, the cells were resuspended in 1 mL of ddH2O and placed on ice for a few minutes to prepare electrically competent cells for gene knockout.

[0069] (5) Knockout fragment transfer and knockout verification Approximately 500 ng of the fusion fragment was added to the prepared electroporation competent cells and incubated on ice for 30 min. Subsequently, the competent cells were added to an electroporation cuvette (Bio-Rad, 2 mm) and electroporated using a MicroPluser electroporator in EC2 mode. After electroporation, 800 μL of LB medium was added, and the bacterial culture was transferred to a 1.5 mL centrifuge tube and incubated at 30°C for approximately 50 min. After incubation, the cells were collected by centrifugation and plated on plates containing 50 μg / mL kanamycin, and incubated at 30°C. Once colonies had grown, the gene knockout was verified using the corresponding primers (Table 3).

[0070] The functions of the gene clusters are shown in Table 4.

[0071] Table 4 Functions of gene clusters

[0072] 4.1.2 Creation of transposon mutants of Pantotheca acuminata strain ZJU23 With Escherichia coli SM10 λpir(pSC123) was used as the donor bacterium, and *Pantotheca acuminata* ZJU23-Rif was used as the recipient bacterium. A Tn5 transposon library of ZJU23 was constructed using a conjugation transfer method. First, donor and recipient strains that grew well on plates were picked and cultured overnight in media containing their respective antibiotics. Then, the OD values ​​of both strains were adjusted to be the same. After centrifugation and washing to remove antibiotics, the donor and recipient strains were resuspended in 100 μL of sterile water and mixed at a 1:1 ratio, then spotted onto a 0.45 μm microporous membrane. This membrane was placed in antibiotic-free LB medium and incubated at 30°C for approximately 6 h for conjugation. After conjugation, the bacterial cells were resuspended in sterile water, diluted, and plated onto a double-antibiotic plate containing kanamycin and rifampin (both at a final concentration of 50 μg / mL). The colonies that grew after incubation were the transposon mutants.

[0073] 4.1.3 Antibacterial activity test As described in Example 1. The test subjects were the metabolic gene cluster knockout strain constructed in "4.1.1" or the transposon mutant constructed in "4.1.2". See the specific results below. Figure 4 .

[0074] Depend on Figure 4 As shown in Figure a, strain ZJU23 only lost its antibacterial activity after the Cluster 7 gene cluster was knocked out. This result ruled out the influence of secondary metabolites on the growth of Bacillus subtilis, indicating that the antibacterial activity of strain ZJU23 may be related to bacterial primary metabolism. Furthermore, this embodiment also used transposon random insertion to screen for four mutants with lost antibacterial activity (…). Figure 4 b) Resequencing analysis of the mutants revealed that multiple mutation sites were concentrated in glucose metabolism and PQQ-related genes. Figure 4 c) region suggests that the antibacterial ability of strain ZJU23 is closely related to PQQ synthesis and glucose oxidation metabolism.

[0075] To further demonstrate the relationship between the antibacterial activity of *Pantheliosy foetida* strain ZJU23 and PQQ synthesis, this example tested the ΔP synthesis after the addition of PQQ. pqq The antibacterial activity of the strain (PQQ synthesis gene cluster knockout mutant) was investigated. Results showed that after adding PQQ, Δ pqq The antibacterial activity of the strain was restored. Figure 5 ).

[0076] In summary, the above results collectively demonstrate that the antibacterial activity of the Pantotheca cum Caulis ZJU23 strain is related to PQQ.

[0077] 4.2 Pantothecin ZJU23 strain inhibits bacterial growth by lowering pH. Because PQQ affects bacterial glucose metabolism pathways and is often associated with the production of organic acids, this embodiment measures the pH environment around the colony. The specific steps are as follows: Add 10 μM bromocresol green indicator (Shanghai Sangon Biotech, catalog number: A354509) to WA medium, and then add 10 μL of OD... 600 =1.0 ZJU23 strain and Δ pqq The bacterial strain was spotted in the center of a plate containing bromocresol green indicator. The color change around the colony was observed. When the area around the colony was yellow, the pH was less than 3.8; when a transitional color from yellow-green to grass green to blue-green was observed, the pH was between 3.8 and 5.4; and when the colony was blue, the pH was greater than 5.4. See below for detailed results. Figure 6 .

[0078] The results show that, containing Δ pqq The culture medium for the mutant was blue; compared to Δ pqq The mutant, the ZJU23 strain, exhibits a yellow-green color in the culture medium surrounding it, indicating an acidic environment. This suggests that the ZJU23 strain may inhibit the growth of Bacillus subtilis by producing acidic substances to lower the pH around the colony. Figure 6 ).

[0079] Furthermore, to demonstrate the correlation between pH environment and the antibacterial activity of strain ZJU23, this example added 2 mL of 10×PBS buffer to WA medium containing strain ZJU23 and Bacillus subtilis, and observed whether the inhibition zone of strain ZJU23 changed. Figure 7 It can be seen that the inhibition zone of strain ZJU23 disappeared after the addition of PBS buffer. This means that the PBS solution neutralized the acidic environment created by the clustered pantothenic bacteria strain ZJU23, resulting in the loss of antibacterial activity of strain ZJU23.

[0080] Furthermore, the above results indicate that Δ pqq The pH of the environment surrounding the mutant colonies was greater than 5.4, but it is unclear whether this is due to Δ pqq The strain was unable to produce PQQ, therefore this example tested the effect of adding PQQ. Δpqq pH of the bacterial colony. Figure 8 It can be seen that as the concentration of PQQ in the culture medium increases, Δpqq The increasing acidification zone around the strain indicates that PQQ has recovered. Δpqq The mutant's acid-producing ability can be restored based on PQQ. Δpqq The antibacterial activity of the mutant was investigated, suggesting that the Pantotheca cum C. ZJU23 strain inhibited bacterial growth by synthesizing PQQ and lowering pH.

[0081] 4.3 The Pantotheca acuminata ZJU23 strain inhibits bacterial growth by producing gluconic acid and lowering the pH. The above experimental results demonstrate that the *Pantotheca cum Caulis ZJU23* strain only exhibits strong antibacterial activity in media with glucose as the carbon source. The *Pantotheca cum Caulis ZJU23* strain inhibits bacterial growth by synthesizing PQQ and lowering the pH. Given that PQQ acts as a coenzyme for membrane-bound glucose dehydrogenase (Gcd) in the periplasmic oxidation of glucose to gluconic acid, but it is uncertain whether gluconic acid is the compound produced by the *Pantotheca cum Caulis ZJU23* strain that lowers the colony pH, this embodiment knocked out genes related to the glucose metabolism pathway in the ZJU23 strain and detected the antibacterial activity, colony pH, and gluconic acid content of the knockout line. The specific steps are as follows: 4.3.1 Construction of glucose metabolism pathway gene knockout lines The scarless knockout method uses the suicide-type plasmid pKD4SacB. Plasmid pKD4SacB is derived from pEX18GM... sacB Gene sequence cloned into plasmid pKD4 Cla This plasmid was obtained by restriction enzyme cleavage at the I restriction site. It contains an R6K replicon, whose replication depends on the expression of the PI protein, and therefore can be detected in *E. coli* S17-1. λpir Replication is initiated in the strain. Information on the glucose metabolism pathway genes to be knocked out in this example is shown in Table 5.

[0082] Table 5. Gene information on glucose metabolism pathways

[0083] The specific knockout steps are as follows: (1) Design and cloning of homologous arms of the target gene: Primers were designed to amplify the upstream and downstream fragments of the target gene. The upstream and downstream fragments were fused using overlap extension PCR technology and cloned into plasmid pKD4SacB. Pvu Site I, transfected into E. coli S17-1 λpir The primers for amplifying the gene are shown in Table 6.

[0084] Table 6 Primer sequences for gene amplification

[0085] (2) Transformation of suicide plasmid into target strain: The constructed suicide plasmid was transformed into the target strain using the electroporation transformation method (see the specific steps in 4.1.1 for preparation of electroporation competent cells and electroporation method) and then plated onto LB plates containing kanamycin.

[0086] (3) Two rounds of homologous recombination screening: The first round of homologous recombination yielded mutants with single crossovers, and the second round of recombination utilized... SacBGene. The first recombinant mutant was confirmed by PCR. Subsequently, the confirmed single colonies were selected and cultured overnight in LB medium. The next day, the bacterial suspension was diluted 1,000 times and spread onto LB plates containing 10% sucrose for reverse selection. The plates were incubated at 25°C for 2-3 days. After single colonies grew on the sucrose plates, single colonies were picked and spread onto LB plates and LB plates containing kanamycin. Colonies that grew normally on LB plates but not on kanamycin plates were the strains that underwent double crossover. Finally, these strains were verified by PCR to obtain the successfully knocked-out, traceless knockout mutants.

[0087] 4.3.2 Detection of antibacterial activity The steps for detecting the antibacterial activity of strain ZJU23 or its mutants on solid culture medium are the same as described above. Specific experimental results can be found in [link to relevant documentation]. Figure 9 .

[0088] 4.3.3 Detection of colony pH The pH detection procedure for colonies of strain ZJU23 or its mutants inoculated on solid plates is the same as described above. For specific experimental results, please refer to [link / reference needed]. Figure 10 .

[0089] Add 1 mL of OD to 100 mL of WA liquid culture medium. 600 = 1.0 ZJU23 strain, loss of antibacterial activity Δ pqq strain, Δ gad1 Δ gad2 strain 、 Δ gad1 Δ gad2 Δ gnuk The strain was cultured at 30℃ and 180 rpm for 1 day, and the pH of the fermentation broth was then measured. Specific experimental results are shown below. Figure 12 .

[0090] 4.3.4 Determination of the growth curve of Bacillus subtilis 3610 Add 1 mL of OD to 100 mL of WA liquid culture medium. 600 =1.0 ZJU23 strain, Δ pqq strain, Δ gad1 Δ gad2 strain 、 Δ gad1 Δ gad2 Δ gnuk The strain was cultured at 30℃ and 180 rpm for 1 day; after centrifugation, 500 μL of supernatant was collected using a sterile filter, and the supernatant was then mixed with OD... 600=1.0 of Bacillus subtilis 3610 was mixed at a volume ratio of 1:1. The growth curve was obtained by measuring the cell optical density (OD) at 30-minute intervals in a BioTek plate reader (Cytation 1). 600 The growth was monitored using nm (100 nm), and the experiment lasted for 24 hours. A growth curve for Bacillus subtilis 3610 was plotted; detailed experimental results can be found in [link to experimental results]. Figure 11 .

[0091] The pH of LB medium was adjusted with gluconic acid, and then 500 μL of OD was inoculated at a volume ratio of 1:1. 600 The growth curve of Bacillus subtilis strain 3610 with an optical density of 1.0 was obtained by measuring cell optical density (OD) at 30-minute intervals in a BioTek plate reader (Cytation 1). 600 The growth was monitored using nm (100 nm), and the experiment lasted for 24 hours. A growth curve for Bacillus subtilis 3610 was plotted; detailed experimental results can be found in [link to experimental results]. Figure 14 .

[0092] Growth curves were monitored by measuring cell optical density (OD600 nm) at 30-minute intervals in a BioTek plate reader (Cytation 1) for 24 hours.

[0093] 4.3.5 Determination of gluconic acid Add 1 mL of OD to 100 mL of WA liquid culture medium. 600 =1.0 ZJU23 strain, Δ pqq strain, Δ gad1 Δ gad2 strain 、 Δ gad1 Δ gad2 Δ gnuk The strain was cultured at 30℃ and 180 rpm for 1 day; after centrifugation, the supernatant was collected using a sterile filter and analyzed by mass spectrometry. Detailed experimental results are available in [link to experimental results]. Figure 13 .

[0094] Depend on Figures 9-10 It was found that only the knockout strain producing the acidified region possessed antibacterial activity, further verifying the relationship between pH and the antibacterial activity of the Pantotheca acuminata ZJU23 strain. Furthermore, only when inoculated with a mutant strain capable of producing gluconic acid (Δ... gad1 Δ gad2 strain 、 Δ gad1 Δ gad2 Δ gnuk Only when the culture medium of the strain (a) did acidification zones and inhibition zones appear, and Δ gad1 Δ gad2 strain、 Δ gad1 Δ gad2 Δ gnuk The inhibition zone of the strain is the same size as that of the wild-type ZJU23 strain, and the acidification zone of the former is even larger than that of the latter.

[0095] from Figure 11 It can be seen that, in the absence of strain ZJU23 or its mutant, the growth curve of Bacillus subtilis strain 3610 is S-shaped, indicating normal growth; with the addition of Δ... pqq After the strain was selected, the growth of Bacillus subtilis was inhibited to some extent in the early stage, but Δ pqq The inhibitory effect of the strain was subsequently broken, no longer affecting the growth of Bacillus subtilis, while Δ gad1 Δ gad2 strain 、 Δ gad1 Δ gad2 Δ gnuk The presence of this strain completely inhibited the growth of Bacillus subtilis strain 3610, with an inhibitory effect consistent with that of the wild-type strain ZJU23. Figure 9 The results suggest that strain ZJU23 inhibits bacterial growth by secreting gluconic acid. Furthermore, pH measurements of the fermentation broth showed that the bacterial solutions with the highest to lowest pH were ordered as follows: Δ pqq >Δ gad1 Δ gad2 >Wild type ZJU23>Δ gad1 Δ gad2 Δ gnuk This trend matches the antibacterial activity of the aforementioned strains. Based on the antibacterial activity test results, strain ZJU23 or its mutants can only inhibit bacterial growth when the pH of the culture medium is <4. Figure 12 ).

[0096] To further demonstrate that the antibacterial acidic substance secreted by strain ZJU23 is gluconic acid, this example also tested the gluconic acid content in different bacterial solutions and the inhibitory effect of gluconic acid on Bacillus subtilis. Figure 13 It can be seen that the bacterial cultures with gluconic acid content from high to low in the fermentation broth are ordered as follows: Δ gad1 Δ gad2 Δ gnuk >Δ gad1 Δ gad2 >Wild type ZJU23>Δ pqq This trend matches the strain's pH and antibacterial activity. Among them, Δ gad1 Δ gad2 Δ gnuk The gluconic acid content in the bacterial culture was as high as 3.49 mg / mL. 。 Figure 14 This demonstrates that only when gluconic acid adjusts the pH of the culture medium to <3 can the growth of Bacillus subtilis be completely inhibited. This result is consistent with... Figure 12 Consistent.

[0097] All the above experimental results indicate that the *Pantotheca acuminata* strain ZJU23 inhibits bacteria by producing gluconic acid and lowering the pH. Simultaneously, it locks... gcd , pqqABCDEF This study identifies key functional genes for high gluconic acid production and enhanced antibacterial activity, providing clear targets for the genetic engineering enhancement of biocontrol strains.

[0098] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, are covered within the scope of protection of the present invention.

Claims

1. Clumping of pantothecin ( Pantoea agglomerans The use of strain ZJU23 or its mutants, or preparations containing strain ZJU23 or its mutants, in inhibiting bacteria or preparing antibacterial products, is characterized by: The ZJU23 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 16174 and deposit date of July 30, 2018.

2. A method for inhibiting bacteria, characterized in that, The bacteria are inhibited by using Pantotheca acuminata strain ZJU23 or its mutants, or by preparations containing said strain ZJU23 or its mutants; said strain ZJU23 is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.16174 and deposit date of July 30, 2018.

3. The method according to claim 2, characterized in that, The OD of the Pantotheca acuminata ZJU23 strain or its mutants 600 =0.8~1.

2.

4. The application according to claim 1 or the method according to claims 2-3, characterized in that, The *Pantotheca acuminata* strain ZJU23 or its mutants inhibit the bacteria by any of the following: producing gluconic acid; synthesizing pyrroloquinoline quinone (PQQ); or lowering the pH.

5. The application according to claim 1 or the method according to claims 2-3, characterized in that, The preparation containing the ZJU23 strain or its mutant also includes a culture medium.

6. The application or method according to claim 5, characterized in that, The culture medium includes any one or more of WA medium, TSB medium, LB medium, LBGM medium, NA medium, M9 medium, KB medium, and R2A medium; preferably WA medium.

7. The application or method according to claim 5, characterized in that, The carbon source of the culture medium includes any one or more of glucose, trehalose, starch, mannose, fructose, maltose, D-arabinose, D-xylose, rhamnose, glycerol, lactose, sucrose, mannitol, dextrin, and melibiose; glucose and D-arabinose are preferred; glucose is more preferred.

8. The application according to any one of claims 1, 4-7, or the method according to any one of claims 2-7, characterized in that, The bacteria are either Gram-negative or Gram-positive.

9. Clumping of pantothecin ( Pantoea agglomerans The application of strain ZJU23 or its mutants, or preparations containing strain ZJU23 or its mutants, in the production of gluconic acid or the preparation of gluconic acid products, is characterized in that... The ZJU23 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 16174 and deposit date of July 30, 2018.

10. The application according to any one of claims 1, 4-9 or the method according to any one of claims 2-8, characterized in that, The mutant of the ZJU23 strain is Δ gad1 Δ gad2 or Δ gad1 Δ gad2 Δ gnuk .

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