Construction and application of a mucin-degrading Lactobacillus gasseri strain and its metallopeptidase expression engineered strain

Abstract

This invention belongs to the field of microbiology, specifically relating to the construction method and application of a strain of *Lactobacillus gasseri* that degrades mucin and its engineered metallopeptidase expression strain. This invention discloses a strain of *Lactobacillus gasseri* (… Lactobacillus gasseri YS1021, deposited at CGMCC (accession number 34140), is a strain that utilizes mucin as a nitrogen source to promote growth and significantly increases the production of metabolites such as lactic acid and acetic acid. Comparative transcriptomics screening identified the key gene RS7445 associated with mucin degradation. Overexpression of this gene in *Lactobacillus gasseri* YS1021 resulted in engineered bacteria exhibiting stronger mucin degradation capabilities and a stronger inhibitory effect on the growth, adhesion, and biofilm formation of *Gardnerella vaginalis*. This strain has potential applications in the preparation of drugs, health products, or compositions for the prevention or treatment of bacterial vaginosis and the maintenance of the female reproductive tract microecological balance.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to the construction method and application of a mucin-degrading Lactobacillus gasseri and its metallopeptidase expression engineered strain. Background Technology

[0002] The female reproductive tract niche is composed of mucosal tissue (epithelial cells), the mucus barrier, and the colonized microbial community, exhibiting physiological dynamic changes. It is generally believed that a dominant lactobacillus microbial community is an important indicator of reproductive tract health, while a significant decrease in lactobacillus and a rapid proliferation of pathogenic bacteria will alter the microecological homeostasis, disrupt the mucus barrier, and trigger inflammation and infection, leading to bacterial vaginosis, which seriously endangers gynecological and reproductive health. Elucidating the interaction mechanisms of the "microbial community (beneficial bacteria, pathogenic bacteria, symbiotic bacteria, etc.) – mucus barrier – epithelial cells" in the reproductive tract niche is crucial for a deeper understanding of the formation process of niche homeostasis and imbalance. It is a prerequisite and foundation for revealing the pathogenesis of diseases such as bacterial vaginosis and developing effective prevention and treatment methods, and has significant research significance and application value for maintaining women's health and reducing pregnancy risks.

[0003] In the reproductive tract microecology, beneficial lactobacilli and pathogenic bacteria engage in a complex competitive interaction, with one rising as the other falls. They utilize different survival strategies to extract nutrients from the reproductive tract environment to gain competitive advantages in terms of quantity and space occupation. Studies suggest that the dominance of beneficial lactobacilli can inhibit the growth, adhesion, and colonization of pathogenic bacteria, maintain the stability of the mucus layer structure and barrier homeostasis, and prevent pathogenic bacteria from invading and infecting. Conversely, the massive proliferation of pathogenic bacteria, represented by Gardnerella vaginalis, can degrade mucin, disrupt the mucus layer skeletal structure, and form pores, allowing them to invade epithelial cells and cause inflammation and infection. The mucus layer serves as both a protective barrier for reproductive tract epithelial cells and a nutrient source and habitat for the microbial community. As a skeletal molecule of the mucus layer barrier, mucin is an important target for degradation and utilization by pathogenic bacteria; while lactobacilli beneficial to mucus layer stability are generally considered to lack the ability to degrade mucin. However, the applicant's previous work found that in a simulated reproductive tract culture system, vaginal lactobacilli (such as Lactobacillus gasseri) can utilize mucin as a nutrient to promote bacterial proliferation, indicating that they possess mucin metabolism capabilities. However, the specific molecular mechanisms of Lactobacillus mucin metabolism and its role and significance in the survival competition with host bacteria remain unclear.

[0004] Therefore, this project aims to use the screened *Lactobacillus gasseri* as the research object, employing comparative transcriptomics to identify key enzymes involved in mucin degradation, verifying the function of these key enzymes through in situ gene expression, and evaluating the impact of enhanced mucin degradation function on the proliferation, metabolism, and competitive interaction of *Lactobacillus gasseri* with *Gardnerella vaginalis*. This invention will lay the foundation for a deeper understanding of the metabolic mechanisms and survival competition strategies of *Lactobacillus gasseri* in its reproductive tract niche, while also providing theoretical support and technical guidance for the screening of probiotics in the reproductive tract. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention provides the following technical solution: The first aspect of the invention is a strain of Lactobacillus gasseri ( Lactobacillus gasseri YS1021 is characterized in that the strain was deposited on April 9, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34140; and the strain overexpresses M13 metallopeptidase RS7445.

[0006] Furthermore, the amino acid sequence of the M13 metallopeptidase RS7445 is shown in SEQ ID NO:8.

[0007] Furthermore, the encoding gene for the M13 metallopeptidase is shown in SEQ ID NO:7.

[0008] Secondly, the use of *Lactobacillus gasseri* in the preparation of medicaments or compositions for the prevention or treatment of bacterial vaginosis is provided, characterized in that the strain is *Lactobacillus gasseri* (…). Lactobacillus gasseri YS1021 was deposited on April 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34140; the strain described overexpresses M13 metallopeptidase RS7445.

[0009] Furthermore, the amino acid sequence of the M13 metallopeptidase RS7445 is shown in SEQ ID NO:8.

[0010] Furthermore, the encoding gene of the M13 metallopeptidase RS7445 is shown in SEQ ID NO:7.

[0011] Thirdly, a genetically engineered strain is provided, characterized in that the strain is an engineered strain 7445OE obtained by overexpressing the M13 metallopeptidase RS7445 gene in Lactobacillus gasseri YS1021 as described in the first aspect through genetic engineering.

[0012] Fourthly, the use of the genetically engineered strains described in the third aspect in the preparation of drugs or health products for promoting the proliferation of Lactobacillus gasseri, inhibiting the growth, adhesion, and biofilm formation of Gardnerella vaginalis.

[0013] The beneficial effects of this invention include: (1) Mucin, as a nitrogen source, can be utilized by various reproductive tract lactobacilli such as Lactobacillus gasseri YS1021, significantly promoting cell proliferation; the addition of mucin can promote the synthesis of lactic acid and acetic acid by Lactobacillus gasseri.

[0014] (2) Transcriptomics methods were used to identify important enzymes involved in the degradation and utilization of mucin in Lactobacillus gasseri YS1021, one of which is RS7445, which belongs to the M13 peptidase family.

[0015] (3) The expression of M13 metallopeptidase RS7445 promotes the synthesis of fatty acids and other metabolites in Lactobacillus gasseri.

[0016] (4) Expression of RS7445 is beneficial to enhance the ability of Lactobacillus gasseri to inhibit the growth, adhesion and biofilm formation of Gardnerella vaginalis. Attached Figure Description

[0017] Figure 1 Growth curves of Lactobacillus gasseri YS1021 in different culture media; Figure 2 Changes in lactic acid and acetic acid content of Lactobacillus gasseri YS1021 cultured in different culture media for 36 h; Figure 3 Statistical graph (A) and volcano plot (B) of differentially expressed genes in Lactobacillus gasseri YS1021 that degrade mucin; Figure 4 The relative expression levels of potential differentially expressed genes in Lactobacillus gasseri YS1021 that degrade mucin; Figure 5 Electrophoresis image of Lactobacillus gasseri YS1021 genomic DNA; Figure 6 Electrophoresis image of the RS7445 gene expression recombinant vector plasmid; Figure 7 Relative transcription levels of genes in engineered strain 7445OE; Figure 8 Growth curves of wild-type strain YS1021 and engineered strain 7445OE; Figure 9 Mucin degradation plate experiment of wild-type strain YS1021 (1) and engineered strain 7445OE (2) Figure 10PCA score of 7445OE engineered bacteria in different culture media; Figure 11 OPLS-DA score (A) and displacement test (B) of 7445OE engineered bacteria in different culture media; Figure 12 Volcano diagram of differential metabolites from different nitrogen sources in engineered bacteria 7445OE; Figure 13 Z-score analysis of differential metabolites from different nitrogen sources in engineered bacteria 7445OE; Figure 14 Correlation chord diagram of differential metabolites in 7445OE engineered bacteria Figure 15 Bubble chart of metabolic pathway enrichment analysis of 7445OE engineered bacteria Figure 16 The viable count, adhesion ability, and biofilm amount of Gardnerella vaginalis (GV) were measured after co-culturing wild-type strain YS1021 and engineered strain 7445OE with Gardnerella vaginalis (GV) to antagonize each other. Detailed Implementation

[0018] The following detailed embodiments further illustrate the concept and technical effects of the present invention to fully understand its purpose, features, and effects. Unless otherwise specified, all methods described are conventional methods. Unless otherwise specified, all materials are available from publicly available commercial sources. The illustrative embodiments and descriptions of the present invention are used to explain the invention and do not constitute an undue limitation thereof. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0019] The specific implementation methods utilize the following biological materials, reagents, and instruments: 1. The strains and plasmids used in the experiment are shown in Table 1.

[0020] Table 1. Strains and Plasmids Among them, the strain of the present invention L. gasseri YS1021 is Lactobacillus gasseri ( Lactobacillus gasseri The specimen was deposited on April 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34140, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0021] 2. The reagents used in the experiment are shown in Table 2.

[0022] Table 2 Reagents 3. The instruments and equipment used in the experiment are shown in Table 3.

[0023] Table 3 Instruments and Equipment 4. Preparation of experimental solutions: 1) MRS liquid culture medium (g / L): peptone 10.0, yeast extract 5.0, K2HPO4 2.0, diammonium citrate 2.0, sodium acetate 5.0, glucose 20.0, Tween 80 1.0, MgSO4·7H2O 0.58, MnSO4·4H2O 0.25, pH 6.2-6.4, sterilized at 121℃ for 15 min.

[0024] 2) MRS solid medium: Add 1.5% agar powder to MRS liquid medium, pH 6.2~6.4, and sterilize at 121℃ for 15 min.

[0025] 3) Basic component B: 0.5% glucose, 0.35% sodium chloride, 0.2% lactic acid, 0.14% potassium hydroxide, 0.1% acetic acid, 0.04% urea, 0.02% calcium hydroxide, and 0.02% glycerol are dissolved in distilled water, the pH is adjusted to 7, sterilized at 121℃ for 15 minutes, and then stored for later use.

[0026] 4) Basic component B+mucin (B+M): Add 1% mucin to B, adjust pH=7, sterilize at 121℃ for 15 minutes and store for later use.

[0027] 5) Basic component B + compound nitrogen source (B+N): Add 0.6% peptone, 0.2% beef powder and 0.2% yeast powder to B, adjust the pH to 7, sterilize at 121℃ for 15 min and store for later use; B+N+M and B+2N are compounded based on the above culture medium.

[0028] 6) LB medium (g / L): yeast extract 5.0, tryptone 10.0, NaCl 10.0, pH 7.0, 121.0℃, sterilized for 15.0 min, solid medium with added agar 15.0.

[0029] 7) 1×TAE solution: Take 20.0 mL of 50×TAE solution and add it to a 1000 mL volumetric flask, then add distilled water to make up to 1000 mL.

[0030] 8) 50% glycerol solution (v / v): Mix 50.0 mL of glycerol with 50.0 mL of distilled water, sterilize at 121℃ for 15 min, and set aside for use.

[0031] 9) GS-GM17 medium: Add 2.5g glycine and 17.1g sucrose to GM17 medium, dissolve them completely, and bring the volume to 100mL. Sterilize by filtration through a 0.22μm filter membrane and store at 4℃.

[0032] 10) Washing buffer I: Dissolve 42.8g sucrose and 2.5mL glycerol in deionized water, bring the volume to 250mL, sterilize for 15min, and store at 4°C.

[0033] 11) S-GM17-MC: Add 0.9g MgCl2·6H2O and 0.02g CaCl2 to 100mL SGM17 medium, dissolve completely, filter through a 0.22μm filter membrane for sterilization, and store at 4°C.

[0034] 12) SGM17: Add 17.1g of sucrose to GM17 medium, dissolve it completely, and bring the volume to 100mL. Sterilize by filtering through a 0.22μm filter membrane and store at 4℃.

[0035] 13) 20% glycine stock solution: Dissolve 20g of glycine in deionized water, bring the volume to 100mL, autoclave at 121℃ for 15min, and store at room temperature away from light.

[0036] 14) MRS-S medium (MRS, 0.3M sucrose): Dissolve 10.27g of sucrose in MRS medium, bring the volume to 100mL, and autoclave at 121℃ for 15min.

[0037] 15) MRS-SM medium (MRSS, 0.1M MgCl2): Add 2.0g MgCl2 to 100ml MRS-S medium.

[0038] 16) Washing Buffer II (0.3M sucrose, 1mM MgCl2): Dissolve 10.27g sucrose and 0.02g MgCl2·6H2O in 100mL deionized water, autoclave at 121℃ for 15min, and store at 4℃.

[0039] 17) 30% PEG 1500: Add 15g of PEG 1500 to deionized water, make up to 50mL, sterilize with a 0.22μm filter membrane, and store at 4℃.

[0040] 18) Phosphate buffer: 30mM NaH₂PO₄, 100mM NaCl, pH=8.0 19) Equilibration buffer: 10mM Tris, 300mM NaCl, 50mM NaH2PO4, 20mM imidazole, pH=8.0 20) Impurity removal buffer: 10mM Tris, 300mM NaCl, 50mM NaH2PO4, 100mM imidazole, pH=8.0 21) Elution buffer: 10mM Tris, 300mM NaCl, 50mM NaH2PO4, 200mM imidazole, pH=8.0 22) Protein electrophoresis buffer: 1000mL system, 3.03g Tris, 14.4g glycine, 1g SDS.

[0041] 23) Fixative: 500 mL ethanol, 100 mL glacial acetic acid, 400 mL water.

[0042] 24) Decolorizing solution: 250mL 95% ethanol, 80mL acetic acid, bring to a final volume of 1000mL.

[0043] 25) Staining solution (Coomassie Brilliant Blue): 100 mL system. 45 mL methanol, 1 mL acetic acid, 0.5 g Coomassie Brilliant Blue R-250.

[0044] 5. The primer sequences used in the experiment are shown in Table 4, and the design was carried out using Primer Premier 5.0 software.

[0045] Table 4 Primer List Example 1: Lactobacillus that can utilize mucin 1) Mucin promotes bacterial growth Activated YS1021 was inoculated into B, B+N, and B+M liquid culture media at an inoculation rate of 2% (v / v). After mixing, the bacterial culture was transferred to 96-well plates. The 96-well plates were sealed with a breathable sealing membrane and placed in a microbial growth curve detection system for static incubation at 37°C for 72 hours to monitor their growth.

[0046] The results are as follows Figure 1 As shown: Growth curves of Lactobacillus gasseri YS1021 in different component culture media are as follows. Figure 1 As shown, the growth curves of Lactobacillus vary in different components and among different Lactobacillus species in the same component. YS1021 exhibits the strongest growth ability in B+N+M medium. When other nitrogen sources (beef meal, peptone, and yeast extract) are already present in the culture medium, the addition of mucin can promote the growth of YS1021.

[0047] 2) Mucin alters strain metabolism The effect of mucin on Lactobacillus metabolism was analyzed using high-performance liquid chromatography (HPLC). The results are as follows: Figure 2As shown, after 14 h, the lactic acid content of Lactobacillus gasseri in B+N+M was significantly increased and higher than that in other components. The content in B+N+M was higher than that in B+N, indicating that the addition of mucin in the presence of a complex nitrogen source can promote the metabolism of Lactobacillus to produce lactic acid.

[0048] Figure 2 The study also showed that the acetic acid content of *Lactobacillus gasseri* generally increased across different components. At 12 h, there was no significant difference in acetic acid content between B+N+M and B+2N, and both were higher than the acetic acid content in the other two components. After 12 h, the acetic acid content increased in all components, with B+2N showing a higher acetic acid content than the other components, and B+N+M showing a higher acetic acid content than B+N. This indicates that adding mucin to a complex nitrogen source can promote the production of acetic acid by *Lactobacillus gasseri*.

[0049] 3) The effect of mucin on the gene expression profile of bacterial strains Major differentially expressed genes analysis: Genes with a differential expression level of Padj < 0.05 & |log2FC| > 0 were statistically analyzed to determine the number of significantly differentially expressed genes in the B+M and B+N groups. During the entire metabolic process of *Lactobacillus gasseri* YS1021, a total of 1027 differentially expressed genes showed significant changes in expression levels, including 521 significantly upregulated genes and 506 significantly downregulated genes. Figure 3 (AB). This indicates that, compared with beef meal, peptone, and yeast powder as nitrogen sources, mucin has a different effect on strain growth.

[0050] Based on previous research reports of potential enzymes involved in mucin degradation, and combined with the above results, we will focus on the following genes that are significantly upregulated during metabolism. GADPH Using genes as internal controls, primers were designed and the transcriptional levels of the following genes were examined and verified after 20 h of culture. Figure 4 ). RS7445 Gene transcription levels were 3.7 times higher than in the control group. RS7445 The gene-encoded protein belongs to the M13 family of metallopeptidases. Because mucin contains a linear peptide backbone and proline, RS7445 The gene-encoded enzyme belongs to the M13 peptidase family, therefore it is inferred that... RS7445 Genes are associated with mucin degradation.

[0051] Example 2: Gene cloning of Lactobacillus metabolizing mucin-related enzymes 1. Extraction of Lactobacillus genomic DNA Will L. gasseriYS1021 was activated for two generations and inoculated into MRS liquid medium, then cultured at 37°C for 18 hours. 500 μL of the bacterial culture was centrifuged at 10,000 rpm for 1 minute, and the supernatant was removed. Lactobacillus genomic DNA was extracted from the bacterial precipitate using a bacterial genomic DNA extraction kit. The concentration of the collected genomic DNA solution was determined using a nucleic acid quantification instrument and stored at -20°C.

[0052] The results are as follows Figure 5 As shown in the figure, the genomic DNA band of the strain is bright and uniform, exceeding 10,000 bp, and its size is consistent with the target band, indicating that the extracted genomic DNA meets the requirements for subsequent experiments. After sequencing, RS7445 The gene sequence is: Its protein sequence is as follows: (SEQ ID NO:8) Example 3: Construction of Lactobacillus gasseri engineered strain 7445OE with RS7445 gene overexpression 1. RS7445 PCR amplification of gene fragments (1) According to L. gasseri YS1021 RS7445 Genetic information and multiple cloning site information of pSIP411 plasmid, design RS7445 The PCR primers (7445OE-F and 7445OE-R) were prepared and sent to a sequencing company for synthesis.

[0053] (2) Using YS1021 genomic DNA as a template, the above primers were used for... RS7445 The PCR amplification of the gene, the PCR reaction system and reaction procedure are shown in Table 5 below: Table 5 PCR reaction system The PCR reaction procedure is shown in Table 6 below: Table 6 PCR reaction procedure 2. Construction of recombinant expression vectors Using restriction endonucleases Nco I and Eco RI pair RS7445 The gene and the empty vector plasmid pSIP411 were double-digested with enzymes, and the reaction system is shown in Table 7 below: Table 7 Double Enzyme Digestion Reaction System Add the reagents to the centrifuge tube according to the dosage in the table, mix gently, centrifuge briefly, and incubate at 37°C for 30 min. The resulting enzyme digestion products are separated and recovered by gel electrophoresis (e.g., Figure 6 (As shown). The enzyme-digested and recovered gene fragments and vector were ligated using ligase. 25 ng of the vector was ligated with 3 times the molar amount of... RS7445 The gene fragments were mixed and the volume was adjusted to 5 µL with deionized water. 5 µL of electroligase reaction buffer and 1 µL of ligase were added, and the mixture was incubated at room temperature for 60 min.

[0054] 3. Preparation of competent Lactobacillus gasseri cells: (1) Add 500 μL of 20% glycine stock solution to 9.5 mL of MRSS medium, and inoculate with 100 μL of overnight activated glycine. L. gasseri YS1021 bacterial culture was incubated at 37℃ until the OD600 nm value reached 0.4 ~ 0.6. (2) Place the cultured bacterial solution into a 10 mL centrifuge tube, centrifuge at 6000 × g and 4℃ for 10 min, and discard the supernatant; (3) Resuspend the bacterial cells in 2 mL Washing buffer II, centrifuge at 6000 × g and 4℃ for 10 min, and discard the supernatant; (4) Repeat the above steps; (5) Resuspend the bacterial cells in 2 mL of 30% PEG-1500, centrifuge at 6000 × g and 4℃ for 10 min, and discard the supernatant; (6) Resuspend the bacterial cells in 200 μL of 30% PEG-1500, and aliquot 40 μL into 1.5 mL centrifuge tubes. Place them in an ice bath for later use or freeze them in liquid nitrogen and store them at -80°C.

[0055] 4. Preparation of genetically engineered bacteria (1) Set the electric shock parameters in the electric converter to 1.5kV, 25uF, and 400Ω; (2) Mix 10 μL of the recombinant expression vector ligation product with 100 μL of... L. gasseri Mix YS1021 competent cells thoroughly, gently tap to mix, avoiding the formation of air bubbles, and inject into a pre-cooled 2mm electroporation cuvette for electroporation transformation. Immediately add 1mL of resuscitation medium MRSSM. (3) Transfer the mixture in the electroporation cup to a 1.5 mL centrifuge tube, incubate on ice for 5 min, and then incubate at 37 °C for 2 h; (4) Take 100 μL of the resuscitation culture medium and spread it on the erythromycin-resistant MRS solid medium. At the same time, the remaining 900 μL of bacterial culture can be centrifuged, and after discarding an appropriate amount of supernatant, take 100 μL of the mixed bacterial suspension and spread it on the corresponding antibiotic MRS solid medium. Incubate at 37°C overnight.

[0056] (5) Pick a single colony, inoculate it in 5.0 μg / mL erythromycin MRS liquid medium and culture it overnight. Extract the plasmid using a high-purity plasmid small-volume extraction kit and verify it by agarose gel electrophoresis.

[0057] The study was conducted using RT-qPCR. RS7445 Gene expression levels after 12 hours of fermentation in wild-type and engineered bacteria. Figure 7 As shown, the transcription level of the overexpressed gene was significantly increased compared to the original strain, indicating that the engineered strain was successfully constructed. The expression level of the RS7445 gene was 3.84 times higher than that of the wild-type YS1021 after overexpression.

[0058] Example 4: Comparison of growth curves between wild-type YS1021 and gene-expression engineered strain 7445OE By monitoring the growth curve, it can be seen that L. gasseri Neither the wild-type YS1021 strain nor the overexpression strain 7445OE could grow in basal medium B. In basal medium with a mixed nitrogen source (B+N), there was no significant difference in growth ability between the wild-type and overexpression engineered strains. In basal medium with mucin (B+M), the overexpression engineered strain 7445OE grew better than the wild-type YS1021. This indicates that adding mucin significantly promotes the proliferation of the engineered strains. The growth curves of the wild-type and engineered strains are shown below. Figure 8 As shown. Plate degradation assays were used to evaluate the ability of the overexpressed strains to utilize mucin. Aminoblack dye specifically binds to the protein under acidic conditions, such as... Figure 9 As shown, L. gasseri YS1021 has a lysis zone, and the lysis zone of the overexpressing engineered bacteria is more distinct, therefore L. gasseriYS1021 has the ability to degrade mucin, and the engineered strains expressed have an even stronger ability to degrade mucin.

[0059] Example 5: Evaluation of the ability of RS7445 overexpressing engineered bacteria to utilize mucin based on metabolomics. Non-targeted metabolomics can be used to identify potential advantageous products from mucin degradation and the intrinsic relationship between these products and overexpressed genes.

[0060] (1) Differential sample quality analysis—Principal component analysis (PCA) The samples were cultured in the control group in basal medium plus a compound nitrogen source. RS7445 The overexpression strain (BN) was cultured in basal medium plus mucin in the experimental group. RS7445 Overexpressing strain (BM), a PCA model was established between the two sample groups to observe the distribution and segregation trend of the two sample groups. In PCA... Figure 10 The results show that the two groups of samples are well separated, with PCA1 at 86.46% and PCA2 at 7.4%, for a cumulative contribution rate of 93.86%. This indicates that the samples cluster well in the PCA plot, and the experimental data have good repeatability and reliability, making them suitable for subsequent analysis.

[0061] (2) Differential sample quality analysis—orthogonal partial least squares discriminant analysis (OPLS-DA) Further through OPLS-DA RS7445 The differences in overexpression of engineered bacteria in different culture media were analyzed using pairwise methods, score plots were generated, and the model was cross-validated 200 times. Figure 11 (AB). In the model, R²Y is 1, indicating strong explanatory power; Q² is 0.999, indicating stable predictive power; the closer this value is to 1, the more reliable the model. A regression line Q² with a Y-intercept less than 0 indicates high reliability of the OPLS-DA model. (Explanation) RS7445 The metabolites of the overexpressed engineered bacteria showed good intra-group reproducibility in both mixed nitrogen source medium and mucin medium, with significant differences between groups.

[0062] (3) RS7445 Analysis of the number of differential metabolites in overexpressed engineered bacteria PCA and OPLS-DA results indicate that PCA and OPLS-DA results RS7445 The overexpressing strains showed significant differences in their ability to utilize mucin and complex nitrogen sources. To further screen the differentially expressed metabolites that degrade mucin and complex nitrogen sources, differential metabolite analysis was performed on 7445OE-B+N and 7445OE-B+M. Metabolite volcanoes were preliminarily screened using |log2FC|>1 (fold change) and P<0.05. Figure 12The results showed 686 significantly differentially regulated metabolites, including 470 significantly upregulated metabolites and 216 significantly downregulated metabolites. This indicates that the addition of mucin has an effect on... RS7445 Overexpression strains have a significant effect.

[0063] (4) RS7445 Analysis of differential metabolites in overexpressed engineered bacteria like Figure 13 As shown, with the metabolite content of group B+N as the baseline, the relatively significant metabolites in group B+M were compared. Among them, the contents of methyl 9-hydroxyoctadecanoate, ricinoleic acid, diethyl oxaloyl propionate, hydroxyisovaleroylcarnitine, polyethylene glycol monolaurate, glycine, lauroyl peroxide, methyl 16-hydroxy-hexadecanoate, prabebeurea, 2,4-difluoro-5-trifluoromethoxyaniline, testosterone phenylpropionate, 2-hydroxypentadecanoic acid, pentadecanoic acid triglyceride, ceramide, L-hocitrulline, chlorproxazine, decanoic acid, diisobutyl adipate, 3-tert-butyl adipic acid, 3-hydroxy-8(Z),11(Z),14(Z)-eicosatotrienoic acid, 3-hydroxymyristic acid, and ranitidine were significantly increased. The relative contents of zanamivir, thiamethoxam sulfoxide, 17-β-(N-tert-butyl-amino-formyl), androst-3, styracin, and L-anserin were decreased. The vast majority of its metabolites were fatty acids, suggesting... RS7445 The mucin products degraded by the engineered bacteria are associated with fatty acids.

[0064] (5) Correlation analysis of differential metabolites In the preliminary analysis, RS7445 The overexpressing engineered bacteria yielded 686 significant metabolites of mucin. These significant metabolites were classified, and pairwise comparisons were performed to analyze the correlations between them. For example... Figure 14 As shown, differentially metabolites can be classified into seven categories: fatty acids, amine compounds, benzene and its derivatives, benzofurans, and organic acids. Correlation analysis and chord diagrams of these metabolites reveal a strong positive correlation among fatty acids, amine compounds, benzene and its derivatives, benzofurans, and organic acids. It is speculated that the degradation of mucin promotes the synthesis of fatty acids and other organic compounds.

[0065] (6) Analysis of differential metabolite enrichment pathways Metabolic pathway enrichment analysis based on the KEGG database can reveal significantly altered metabolic pathways, thus contributing to the interpretation of biological phenotypes. In this analysis, metabolic pathways with a p-value < 0.05 were defined as those with significantly enriched differential metabolites. Fatty acid metabolism pathway, secondary metabolism pathway, biotin metabolism pathway, and flavonoid synthesis pathway showed significant expression (…). Figure 15 ).illustrate RS7445Overexpressing bacteria degrade mucin and convert it into fatty acids. In the ecological niche, fatty acids promote mucin secretion, and mucin secretion is beneficial to the growth and colonization of Lactobacillus gasseri.

[0066] Example 6: Evaluation of the antagonistic effect between RS7445-expressing engineered bacteria 7445OE and Gardnerella vaginalis GV-24 (1) Detection of viable Gardnerella vaginalis count after antagonism of engineered bacteria 7445OE YS1021, 7445OE, and GV-24 were inoculated at the same amount into 200 μL of B+M liquid medium and anaerobically cultured in 96-well plates at 37°C for 48 h. After serial dilution, they were plated onto blood agar plates containing vancomycin and polymyxin, and counted after 48 h of incubation.

[0067] (2) Determination of the adhesion ability of Gardnerella vaginalis after antagonism of engineered bacteria 7445OE Plate preparation: Dissolve mucin in phosphate-buffered saline (PBS) (pH 7.4) to a final concentration of 10 mg / mL; add 100 μL of mucin solution to a Nunc Maxisorp 96-well plate and incubate overnight (12 h) at 4°C for fixation; wash twice with 200 μL PBS to remove excess substrate; add 150 μL of 2% (w / v) bovine serum albumin (BSA) and block at 37°C for 2 h; wash twice with 200 μL PBS to remove excess BSA.

[0068] Adhesion ability test: YS1021, 7445OE, and GV-24 were cultured to the logarithmic growth phase. Equal volumes of GV, GV-YS1021, and GV-7445OE were taken, washed twice with 3 mL PBS, and resuspended in PBS until the final OD600 nm = 0.25. 100 μL of the suspension was taken and plated for counting, recorded as Nt. 100 μL of the suspension was added to a 96-well plate and incubated at 37 °C for 1 h. The bacterial suspension was removed with a pipette and washed twice with 200 μL PBS. 200 μL of 0.05% Triton X-100 solution was added and the plate was shaken at room temperature for 1 h. 100 μL of the suspension was diluted and plated for counting, recorded as Ns. The adhesion rate (%) was calculated as (2 Ns / Nt) × 100%.

[0069] (3) Detection of Gardnerella vaginalis biofilm after antagonism of engineered bacteria 7445OE YS1021, 7445OE, and GV-24 were inoculated into 200 μL of B+M liquid medium at the same inoculum and anaerobically cultured in 96-well plates at 37°C for 48 h. The bacterial culture was discarded, and the wells were slowly rinsed with PBS to remove airborne bacteria. The wells were air-dried for 60 min, and 200 μL of 0.1% (w / v) gentian violet was added to stain the biofilm for 30 min. Excess stains were gently washed with 200 μL of PBS, and the wells were air-dried for 5 min. The gentian violet was dissolved in 200 μL of 33% (v / v) acetic acid solution. The absorbance at 595 nm was measured using a microplate reader.

[0070] The antagonistic results of wild-type Lactobacillus gasseri strain YS1021, engineered strain 7445OE, and Gardnerella vaginalis GV-24 are as follows: Figure 16 As shown, YS1021 has a significant antagonistic effect on Gardnerella vaginalis. When engineered strain 7445OE is co-cultured with Gardnerella vaginalis, the number of viable Gardnerella vaginalis bacteria is significantly reduced. YS1021 inhibits the adhesion ability of Gardnerella vaginalis, and engineered strain 7445OE has a better antagonistic inhibitory effect. The biofilm amount of Gardnerella vaginalis GV-24 in the control group was 1.73. YS1021 inhibits the formation of Gardnerella vaginalis GV-24 biofilm, and engineered strain 7445OE has a better antagonistic inhibitory effect, with a biofilm amount of only 1.1.

[0071] The embodiments described above are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

Claims

1. A strain of Lactobacillus gasseri ( Lactobacillus gasseri YS1021, characterized in that, The strain was deposited on April 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34140; the strain highly expresses M13 metallopeptidase RS7445.

2. The strain according to claim 1, characterized in that, The amino acid sequence of the M13 metallopeptidase RS7445 is shown in SEQ ID NO:

8.

3. The strain according to claim 2, characterized in that, The encoding gene for the M13 metallopeptidase RS7445 is shown in SEQ ID NO:

7.

4. The use of a strain of *Lactobacillus gasseri* in the preparation of a medicament or composition for the prevention or treatment of bacterial vaginosis, characterized in that, The strain is *Lactobacillus gasseri* ( Lactobacillus gasseri YS1021 was deposited on April 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34140; the strain described overexpresses M13 metallopeptidase RS7445.

5. The application according to claim 4, characterized in that, The amino acid sequence of the M13 metallopeptidase RS7445 is shown in SEQ ID NO:

8.

6. The application according to claim 4, characterized in that, The encoding gene for the M13 metallopeptidase RS7445 is shown in SEQ ID NO:

7.

7. A genetically engineered strain, characterized in that, The strain is an engineered strain obtained by overexpressing the M13 metallopeptidase RS7445 gene in Lactobacillus gasseri YS1021 as described in claim 1 through genetic engineering, and the amino acid sequence of the M13 metallopeptidase RS7445 is shown in SEQ ID NO:

8.

8. The genetically engineered strain according to claim 7, characterized in that, The encoding gene for the M13 metallopeptidase RS7445 is shown in SEQ ID NO:

7.

9. The use of the genetically engineered strain according to claim 7 or 8 in the preparation of a drug or health product for promoting the proliferation of Lactobacillus gasseri, inhibiting the growth, adhesion and biofilm formation of Gardnerella vaginalis.

10. The use of Lactobacillus gasseri as described in claim 1 in the preparation of a medicine or health product for maintaining or improving the microecological balance of the female reproductive tract.

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