CN-122024864-A - Acquisition method and application of lactobacillus crispatus specific metabolic substrate, and application of specific CAZyme gene and PUL

CN122024864ACN 122024864 ACN122024864 ACN 122024864ACN-122024864-A

Abstract

The invention belongs to the technical fields of bioengineering and microbiology, and particularly relates to a method for acquiring a specific metabolic substrate of lactobacillus crispatus, application of the specific metabolic substrate and application of a specific carbohydrate active enzyme CAZyme gene and polysaccharide utilization site (PUL). The genome of the lactobacillus crispatus and the genome of the lactobacillus crispatus are obtained from a public database, the genome of the lactobacillus crispatus and the genome of the lactobacillus crispatus are subjected to gene annotation, a CAzyme gene special for the lactobacillus crispatus, a PUL gene and other genes with metabolic functions are found by combining a random forest model and a KEGG metabolic pathway diagram, a substrate which can only be metabolized by the lactobacillus crispatus is found based on the genome difference of the two bacteria, and the capability of the obtained substrate to promote the growth of the lactobacillus crispatus and inhibit the growth of the lactobacillus crispatus is evaluated through experiments. The substrate promotes the growth and reproduction of lactobacillus crispatus, so that the substrate has higher ratio in the vaginal microecology, thereby protecting the vagina and reducing the reproduction of other unfavorable bacteria. Improving the success rate of auxiliary reproduction.

Inventors

SONG TAO
LI MING
Xue Qidi
Zhou Taiyu
WANG YITONG
TIAN JUN
XIE YIRAN

Assignees

大连医科大学

Dates

Publication Date: 20260512
Application Date: 20260130

Claims (8)

1. A method for obtaining a specific metabolic substrate of Lactobacillus crispatus is characterized by comprising the following steps, Step 1, acquiring genome data of multiple strains of two lactobacillus including lactobacillus crispatus and lactobacillus inertia from a public database, and preprocessing; The method comprises the steps of 2, carrying out gene annotation on the preprocessed genome data, namely carrying out annotation on carbohydrate active enzyme CAZyme genes and polysaccharide utilization sites PUL of the genome, and carrying out genome-wide function annotation on the genome, wherein PUL is a gene cluster related to specific carbohydrate metabolism in the genome of bacteria, and a carbohydrate substrate which can be metabolized by PUL is attached to annotation results; Step 3, constructing a feature matrix representing the difference between the genes background of the lactobacillus crispatus and the lactobacillus inertia based on the annotation information of the carbohydrate active enzyme CAZyme genes and the polysaccharide utilization sites PUL in the step 2, forming a gene background difference heat map based on the feature matrix, training the feature matrix by using a random forest algorithm, generating the feature importance ranking of the difference CAZyme genes and PUL of two lactobacillus by using a random forest model, combining the feature importance ranking, comparing the gene background difference heat map, screening a plurality of CAZyme genes or PUL, and considering the CAZyme genes or PUL as CAZyme genes or PUL which are special to the lactobacillus crispatus but not to the lactobacillus inertia if the screening condition is met; Step 4, screening to obtain a candidate saccharide substrate set which can be specifically metabolized by the lactobacillus crispatus based on the CAzyme gene, PUL and KEGG metabolic pathway which are found to be unique to the lactobacillus crispatus and are not possessed by the inert lactobacillus crispatus in the step 3; Step 5, constructing a culture medium containing the specific saccharide substrate, setting a standard MRS culture medium and a sugar-free culture medium as a control, respectively inoculating lactobacillus crispatus and inert lactobacillus serving as the control to different culture mediums, culturing under the conditions of 37 ℃ and anaerobism, measuring the OD value of bacterial liquid at regular intervals, drawing a growth curve, verifying the metabolic capability of lactobacillus crispatus on the specific substrate, and if the lactobacillus crispatus is in a stable phase in the specific substrate culture medium and the OD value of the lactobacillus crispatus is obviously higher than the OD value (P < 0.05) in the sugar-free culture medium, indicating that the substrate can be effectively metabolized by the lactobacillus crispatus, and updating a candidate saccharide substrate set which can be specifically metabolized by the lactobacillus crispatus to obtain a final metabolic substrate set.
2. The method of claim 1, wherein the public database comprises an NCBI genomic database and wherein the preprocessing of step 1 comprises removing low quality, incomplete genomic sequences.
3. The method according to claim 1, wherein the genome is annotated with the carbohydrate-active enzyme CAZyme gene and the polysaccharide-utilizing site PUL, the HMMER and DIAMOND tools are used in combination, the annotation database comprises dbCAN HMMdb, CAZyDB and dbCAN-sub, the sequence alignment method is used for sequence alignment between the bacterial genome and the database genes, and the KEGG orthologous gene group KO number, enzyme classification number EC and KEGG reaction number of the genes are obtained, so that genes unique to single lactobacillus and genes shared by a plurality of lactobacillus can be mapped to the KEGG metabolic pathway based on the above information.
4. The method according to claim 1, wherein in step 3, each CAZyme family and PUL is used as a characteristic unit, each strain of Lactobacillus crispatus and Lactobacillus crispatus is used as a sample unit, the presence of the characteristic is 1 and the absence is 0 in the genome of each strain of Lactobacillus crispatus and Lactobacillus crispatus, the characteristic matrix corresponds to a grid pattern, the horizontal axis is the strain of each bacterium, the vertical axis is each gene, each grid of the grid pattern has a corresponding strain and gene, the genome of the strain has the gene is 1, no gene is 0, and when CAZyme genes or PULs specific to Lactobacillus crispatus and not specific to Lactobacillus crispatus are selected, if a CAZyme gene or PUL exists in more than 70% of the genome of the strain of Lactobacillus crispatus and more than 90% of the genome of the strain of Lactobacillus crispatus does not have the gene or PUL, the gene or PUL is regarded as a CAZyme gene or PUL specific to Lactobacillus crispatus and not have the inert Lactobacillus crispatus.
5. The method of claim 1, wherein in step 3 the KEGG metabolic pathway map is a map file of the target metabolic pathway map obtained directly from the KEGG website, including but not limited to map00010 glycolysis/gluconeogenesis, map00500 starch and sucrose metabolism, map02010 ABC transporter pathway maps, and based on the result of genome-wide functional annotation of both bacterial genomes in step 2, labeling the KEGG metabolic pathway maps according to whether the Lactobacillus crispatus and the Lactobacillus crispatus have the corresponding genes, labeling the Lactobacillus crispatus specific genes as green, labeling the Lactobacillus crispatus specific genes as blue, labeling the two bacteria consensus genes as two colors for intuitively distinguishing and defining the specific genes and specific metabolic pathway of Lactobacillus crispatus, and the step of labeling in the KEGG metabolic pathway map is used to identify key metabolic nodes specific or enriched to Lactobacillus crispatus at the metabolic pathway level and correlate with the CAZyme genes and PUL differential features to support the candidate substrates for subsequent screening and verification.
6. The method according to claim 1, wherein the substrate concentration is set to be 5,10,20 g/L when the medium of the specific saccharide substrate is prepared in the step 5, the basal medium is MRS medium (original carbon source is removed), the OD value of the bacterial liquid in the medium is measured by an enzyme-labeled instrument in the culture process to reflect the growth and propagation condition of bacteria, the measurement wavelength is 600 nm, the culture time is 24 hours and the measurement interval is 6 hours, the growth curve is drawn by GRAPHPAD PRISM software, and the metabolic capacity of the lactobacillus crispatus and the inert lactobacillus on the substrate and the growth delay time, logarithmic phase growth rate and stationary phase OD value of the lactobacillus in the specific substrate medium and the reference medium are judged, if the lactobacillus crispatus is in stationary phase in the specific substrate medium and the OD value of the lactobacillus crispatus is significantly higher than the OD value (P < 0.05) in the sugarless medium, the substrate can be effectively metabolized by the lactobacillus crispatus.
7. The application of the lactobacillus crispatus specific CAZyme gene and the PUL is characterized in that a substrate which can be specifically metabolized by the lactobacillus crispatus is selected based on the gene or the PUL and is used for preparing a product for improving the vaginal microecology, the lactobacillus crispatus specific CAZyme gene and the PUL are identified by the method of the step 3 in the claim 1, the specific CAZyme gene and the PUL comprise, but are not limited to, GH36 (glycoside hydrolase family 36) genes (aga, 3.2.1.22), GH13_29 (glycoside hydrolase family 13_29) genes (treC, 3.2.1.93), GH1 (glycoside hydrolase family 1) genes (pbg, pbg6, 3.2.1.86), PUL0048 (containing GH 13_29) and PUL0088 (containing GH 36), and metabolic auxiliary preparations containing enzymes corresponding to the genes.
8. The application of the specific metabolic substrate of the lactobacillus crispatus is characterized by being used for preparing a product for improving the success rate of an assisted reproduction technology, wherein the specific metabolic substrate of the lactobacillus crispatus is obtained by the method of any one of claims 1 to 6, and the product is a vaginal microecological regulator containing the substrate, and the product can promote the growth of the lactobacillus crispatus in the vagina and inhibit the growth of inert lactobacillus through supplementing the substrate, so that the vaginal flora structure is improved, and the ART success rate is further improved.

Description

Acquisition method and application of lactobacillus crispatus specific metabolic substrate, and application of specific CAZyme gene and PUL Technical Field The invention belongs to the technical fields of bioengineering and microbiology, and particularly relates to a method for acquiring a specific metabolic substrate of lactobacillus crispatus, application of the specific metabolic substrate, and application of a specific CAZyme gene and PUL. Background Assisted reproductive technologies (assisted reproductive technology, ART) are a class of technologies that aid in the pregnancy of sterile couples by medical means. Vaginal microecology has been shown to play a key role in female reproductive health and ART outcome. The vaginal flora with Lactobacillus crispatus (Lactobacillus crispatus) as the dominant is usually accompanied by lower vaginal pH, less bacterial vaginitis (bacterial vaginosis, BV) and higher success rate of pregnancy, while the flora with the dominant strain is more likely to be accompanied by BV recurrence and increased risk of sexually transmitted pathogen infection due to the weaker protective effect of the inert Lactobacillus (Lactobacillus iners) on host epithelium, and is associated with bad outcomes such as abortion, ART graft failure, etc. It is considered that metabolic enzymes such as carbohydrate-active enzymes play an important role in the growth and reproduction of bacteria, and the addition of a specific metabolic substrate for Lactobacillus crispatus to selectively promote the growth of such bacteria may improve the effect and success rate of ART, and thus, the selective promotion of the colonization and growth of Lactobacillus crispatus in the vaginal environment has become an important direction for current micro-ecological regulation. In recent years, carbohydrate-active enzymes (cazymes) and their associated polysaccharide utilization sites (polysaccharide utilization loci, PUL) have been demonstrated to determine the ability of microorganisms to utilize different carbohydrate substrates. The CAZyme of the lactobacillus vaginalis can not only provide necessary energy sources for the CAZyme by decomposing glycogen and other substrates to produce monosaccharides, but also can produce lactic acid and short-chain fatty acid by taking the CAZyme as raw materials to maintain the acidic environment of the vagina. Therefore, if a specific saccharide substrate that can be preferentially utilized by lactobacillus crispatus, but not or less readily utilized by lactobacillus crispatus can be obtained, it is expected that the growth and colonization of lactobacillus crispatus can be selectively promoted by supplementing the substrate, thereby improving the vaginal flora structure and improving ART outcome. Systematic study of the differences between the inert lactobacillus and the lactobacillus crispatus at the CAZyme gene level is the key to screening the prebiotics required for the specific metabolic pathway of lactobacillus crispatus. The existing strategies for improving the vaginal microecology mainly comprise exogenous lactobacillus preparation supplement and broad-spectrum prebiotic supplement, but the schemes often have difficulty in specifically improving the relative abundance of lactobacillus crispatus in a complex vaginal microenvironment, have limited inhibition effect on non-ideal lactobacillus such as inert lactobacillus, and more importantly, the existing strategies generally cannot give a verifiable evidence chain of which substrate or substrate can be selectively utilized by lactobacillus crispatus and lack systematic knowledge of the selective action relationship between specific substrate and specific strain. The development of bioinformatics provides a new means for the above problems, and a variety of gene function annotation tools have been used to resolve metabolic characteristics of lactobacillus. Of these, dbCAN (carbohydrate enzyme gene annotation software) is a tool specific for identifying carbohydrate-active enzymes (CAZyme) in the genome, and protein sequences and PULs can be annotated according to the characteristic domains. eggNOG Gene annotation software is a common homologous gene alignment and function annotation tool, which can perform function annotation based on the existing homologous gene genome, and the annotation result can be mapped to KEGG metabolic pathways, so that the function of a specific gene can be understood by placing the specific gene in the macroscopic metabolic pathway network background. However, at the genomic analysis level, the functions of software such as dbCAN, eggNOG and the like are distributed and universal, each of which only provides annotation information of a certain dimension, and a standardized and systematic professional analysis flow is lacking in the aspect of comparative analysis of carbohydrate metabolism differences among different lactobacillus species. At present, systematic mining of the CAZyme gene diff