Inducible plasmid self-destruction assisted recombination

Abstract

The present invention provides a circular DNA vector that can be used to introduce specific mutations in a target region of a host cell. The present invention further provides methods of using the circular DNA vector to generate engineered host cells. The circular DNA vector and methods can be used to study gene function and to generate cells that produce recombinant gene products.

Description

Technical Field

[0001] This invention provides a circular DNA vector that can be used to introduce specific mutations into target regions of host cells. The invention further provides a method for generating engineered host cells using the circular DNA vector. The circular DNA vector and method can be used to study gene function and generate cells that produce recombinant gene products. Background Technology

[0002] Lactobacillus has been widely used as a probiotic and is increasingly being studied as a delivery vector for medically relevant recombinant proteins to mucosal surfaces. In contrast, genetic tools (especially mutagenesis) for studying and enhancing probiotic activity have been poorly developed. Traditional genome engineering methods rely heavily on bacterial transformation efficiency. This limitation can be overcome with conditional replication plasmid-assisted recombination engineering, such as plasmids containing the pWV01 thermosensitive origin of replication, but the latter may have a limited host range. Therefore, there is a need to develop more flexible and efficient genome editing strategies to better understand and apply these health-promoting microorganisms. Summary of the Invention

[0003] A first aspect of the present invention provides a circular DNA vector comprising:

[0004] (a) An optional marker gene sequence, wherein the marker gene sequence is operatively linked to a first promoter sequence.

[0005] (b) a multiple cloning site, wherein the multiple cloning site optionally includes a gene-targeting sequence.

[0006] (c) A sequence encoding a site-specific recombinase, wherein the sequence is operatively ligated to a second promoter sequence, wherein the second promoter is inducible.

[0007] (d) Copy the subsequence.

[0008] (e) Two target sites for the site-specific recombinase.

[0009] The vector comprises a first region flanked on each side by one of the target sites for the site-specific recombinase, and wherein the region comprises (a) and (b), provided that (c) and (d) are not within the first region.

[0010] The circular DNA vector can be used as a targeting vector for the targeted integration of (gene) sequences into the genome of a host through homologous recombination. The circular DNA vector is particularly useful for gene targeting in host cells that are generally known to be difficult to transform, such as species of Lactobacillus and Bifidobacteria. According to the method of the invention, the circular DNA vector is particularly useful for gene targeting.

[0011] Therefore, in a second aspect of the invention, a method for introducing recombination between a circular DNA vector and a host cell genomic target region is provided, the method comprising the steps of:

[0012] (i) Introducing a circular DNA vector containing a gene-targeting sequence according to the invention into a host cell, wherein the target sequence comprises a flanking sequence containing at least about 200 consecutive nucleotides having at least 80% sequence identity with a corresponding region of the target region of the host cell genome.

[0013] (ii) Inducing the expression of a site-specific recombinase encoded by the circular DNA vector, and allowing site-specific recombination between target sites of the site-specific recombinase to produce a first circular DNA product comprising (a) and (b) and a second circular DNA product comprising (c) and (d).

[0014] (iii) Selecting a host cell, wherein the first circular DNA product comprising (a) and (b) is integrated into the target region of the genome via a first single crossover homologous recombination event between the flanking sequence of the target sequence and the target region of the host cell genome, and

[0015] (iv) Select a host cell in which (a) has been excised from the genome of the host cell obtained in (iii) by a second homologous recombination event between the flanking sequence of the target sequence and the target region of the genome.

[0016] In a third aspect, the present invention provides a method for generating host cells with mutations in target genes of the host cell genome, the method comprising the steps of:

[0017] (i) Introducing a circular DNA vector containing a gene-targeting sequence according to the invention into a host cell, wherein the target sequence comprises flanking sequences containing at least about 200 consecutive nucleotides having at least 80% sequence identity with a corresponding region of a target gene in the host cell genome.

[0018] (ii) Inducing the expression of a site-specific recombinase encoded by the circular DNA vector, and allowing site-specific recombination between target sites of the site-specific recombinase to produce a first circular DNA product comprising (a) and (b) and a second circular DNA product comprising (c) and (d).

[0019] (iii) Selecting a host cell, wherein the first circular DNA product comprising (a) and (b) is integrated into the target region of the genome via a first single crossover homologous recombination event between the flanking sequence of the target sequence and the target region of the host cell genome, and (iv) Selecting a host cell, wherein (a) has been excised from the genome of the host cell obtained under (iii) via a second homologous recombination event between the flanking sequence of the target sequence and the target region of the genome, and wherein the host cell contains a mutation in the target region.

[0020] In a fourth aspect, the present invention provides a method for generating host cells in which target genes in the host cell genome have lost their function, the method comprising the steps of:

[0021] (i) Introducing a circular DNA vector containing a gene-targeting sequence according to the invention into a host cell, wherein the target sequence comprises flanking sequences containing at least about 200 consecutive nucleotides having at least 80% sequence identity with a corresponding region of a target gene in the host cell genome.

[0022] (ii) Inducing the expression of a site-specific recombinase encoded by the circular DNA vector, and allowing site-specific recombination between target sites of the site-specific recombinase to produce a first circular DNA product comprising (a) and (b) and a second circular DNA product comprising (c) and (d).

[0023] (iii) Selecting a host cell, wherein the first circular DNA product comprising (a) and (b) is integrated into the target region of the genome via a first single crossover homologous recombination event between the flanking sequence of the target sequence and the target region of the host cell genome, and (iv) Selecting a host cell, wherein (a) has been excised from the genome of the host cell obtained in (iii) via a second homologous recombination event between the flanking sequence of the target sequence and the target region of the genome, and wherein the host cell contains the target gene and is thus rendered nonfunctional.

[0024] In a fifth aspect, the present invention provides a method for generating host cells with functional gain in target genes of the host cell genome, the method comprising the steps of:

[0025] (i) Introducing a circular DNA vector containing a gene-targeting sequence according to the invention into a host cell, wherein the target sequence comprises flanking sequences containing at least about 200 consecutive nucleotides having at least 80% sequence identity with the target gene of the host cell genome.

[0026] (ii) Inducing the expression of a site-specific recombinase encoded by the circular DNA vector, and allowing site-specific recombination between target sites of the site-specific recombinase to produce a first circular DNA product comprising (a) and (b) and a second circular DNA product comprising (c) and (d).

[0027] (iii) Selecting a host cell, wherein the first circular DNA product comprising (a) and (b) is integrated into the target region of the genome via a first single crossover homologous recombination event between the flanking sequence of the target sequence and the target region of the host cell genome, and (iv) Selecting a host cell, wherein (a) has been excised from the genome of the host cell obtained in (iii) via a second homologous recombination event between the flanking sequence of the target sequence and the target region of the genome, and wherein the host cell contains functional gain of the target gene.

[0028] In a sixth aspect, the present invention provides a method for preparing host cells expressing recombinant polypeptides, the method comprising the steps of:

[0029] (i) Introducing a circular DNA vector containing a gene-targeting sequence according to the invention into a host cell, wherein the target sequence comprises flanking sequences comprising at least about 200 consecutive nucleotides having at least 80% sequence identity with a corresponding region of the target region of the host cell genome, and wherein the gene-targeting sequence encodes a recombinant polypeptide.

[0030] (ii) Inducing the expression of a site-specific recombinase encoded by the circular DNA vector, and allowing site-specific recombination between target sites of the site-specific recombinase to produce a first circular DNA product comprising (a) and (b) and a second circular DNA product comprising (c) and (d).

[0031] (iii) Selecting a host cell, wherein the first circular DNA product comprising (a) and (b) is integrated into the target region of the genome via a first single crossover homologous recombination event between the flanking sequence of the target sequence and the target region of the host cell genome, and

[0032] (iv) Select a host cell in which (a) has been excised from the genome of the host cell obtained in (iii) by a second homologous recombination event between the flanking sequence of the target sequence and the target region of the genome.

[0033] A further aspect of the invention provides a recombinant host cell obtained by any of the methods of the invention.

[0034] In a further aspect, the present invention provides the use of the circular vector of the present invention for introducing gene sequences into the host cell genome.

[0035] In a further aspect, the present invention provides the use of the circular carrier of the present invention for increasing tissue adhesion of host cells. Attached Figure Description

[0036] Figure 1 IPSD strategy for bacterial recombination. a, Schematic diagram of inducible plasmid self-destruction. A vector was constructed in which the replicon and antibiotic resistance gene are separated by two oriented six fragments. After the addition of an inducer and β-mediated recombination, the vector loses function due to replicon excision. Rep, replicon; Pro, controlled expression promoter; Rec, recombinase; six: two oriented six target sequence sites; Ar, antibiotic resistance gene; MCS, multiple cloning site. b, Schematic diagram of IPSD-assisted bacterial recombination engineering, including gene deletion, insertion, and substitution (indicated by asterisks). After recombination, the proportion of colonies containing episomal vectors decreases (blue arrows), while the proportion of colonies with integrated DNA fragments via single crossover increases (red arrows). Single crossover mutant colonies can be screened by PCR, and double crossover clones can be selected by anti-selection. A and B indicate the homologous ends flanking the target genes. Target genes on chromosomes are represented by pink rectangles. c. Lactobacillus gasseri DSM 14869 strain grown on MRS agar plates supplemented with 10 μg / ml chloramphenicol and with or without 100 ng / ml SppIP.

[0037] Figure 2

[0038] Physical map of the IPSD vector pINTZrec. Cmr, chloramphenicol resistance gene; Rec, recombinase gene β.

[0039] Figure 3 IPSD-assisted gene deletions and insertions in Lactobacillus. a, b, The Lactobacillus gasseri DSM 14869upp gene was deleted, and the deletion mutant was verified by PCR using primer pairs upperft-F and upperright-R. c, Compared with WT, the Lactobacillus gasseri DSM 14869upp mutant showed tolerance to 100 μg / ml 5-FU.

[0040] Figure 4

[0041] Indicated Lactobacillus strains were produced on MRS agar plates supplemented with 10 μg / ml chloramphenicol and with or without 100 ng / ml SppIP. (a) Lactobacillus paracasei BL23, (b) Lactobacillus acidophilus ATCC 4356, (c) Lactobacillus plantarum NL42.

[0042] Figure 5

[0043] The upp gene in Lactobacillus gasseri DSM 14869 was deleted using IPSD-assisted recombination engineering.

[0044] (a) The upp-Cmr fragment was single-crossover integrated into the chromosome of *Lactobacillus gasseri* DSM 14869. Colony PCR was performed using primers pIrec-F and pIrec-R to detect the intact plasmid pINTZrec-upp. Ten colonies without a corresponding band were designated as candidate single-crossover clones and marked "+". (b) Single-crossover events in the ten candidate clones were further confirmed by DNA extraction and PCR using primers uppleft-F and pIrecSC-R (top gel) or pIrecSC-F and uppright-R (bottom gel). Correct clones were marked "+", and indeterminate clones were marked "?". (c) Colony PCR was performed using primers uppseq-F and uppseq-R to detect upp double-crossover deletion mutants selected on SDM medium supplemented with 5-FU. M, GeneRuler TM 1kb DNA ladder; P, DNA of Lactobacillus gasseri DSM 14869 containing plasmid pINTZrec-upp; WT, DNA of wild-type Lactobacillus gasseri DSM 14869.

[0045] Figure 6

[0046] The inducible promoters used to drive recombinase expression were ineffective in some strains. *Lactobacillus sakei* NC03 strain (a) and *Lactobacillus rhamnosus* GG strain (b) were grown on MRS agar plates supplemented with 10 μg / ml chloramphenicol under conditions with or without 100 ng / ml SppIP.

[0047] Figure 7

[0048] Schematic diagram of the EPS gene cluster (A) and protein domains of N506_1778 (B) and N506_1709 (C) in *Lactobacillus gasseri* DSM 14869. (A) shows the EPS gene cluster (N506_0396 to N506_0411) located at nucleotide numbers 379,930–394,580 in the genomic sequence (CP006803). Enzyme and gene names were annotated by BLASTP analysis using strains *Lactobacillus gasseri* ATCC 33323 and *Lactobacillus gasseri* CECT5714 (Marcotte et al. 2017). (B) The N506_1778 protein includes a YSIRK signal sequence at the N-terminus and a Gram-positive LPxTG (LPQTG) motif at the C-terminus. The repeating region consists of two MucBP-like domains. The (C)N506_1709 protein comprises an N-terminal YSIRK signal sequence and a C-terminal LPQTG motif. The repeat region contains three distinct Rib / alpha-like repeats, one of which is a partial repeat. The N-terminus (approximately 1–900 aa) shows no similarity to other proteins in the database, while the C-terminus (approximately 900–1456 aa) shows a lower percentage of identity (34–48%) with the C-termini of proteins from *Lactobacillus johnsonii* and *Lactobacillus gasseri* (accession numbers WP_095670316, QYS15157, WP_061400034, OYS08635, OYS05727, OUL52955, and AHA97914). Gene and protein structures are not shown to scale.

[0049] Figure 8

[0050] (A) Transmission electron micrographs of wild-type (WT), EPS mutant (ΔEPS), and complement strains of *Lactobacillus gasseri* DSM 14869. A dense EPS layer was observed in the WT, while surface EPS production was significantly reduced in the EPS mutant. The EPS complement strain restored EPS levels to those of the WT. Scale bar = 200 nm. (B) Comparison of EPS layer thickness in the WT, ΔEPS, and EPS complement strains. Relative thickness is shown as a percentage relative to the WT (set to 100%). Thickness was assessed in 10 cells per group, and the mean ± SD of thickness for each cell was determined.

[0051] Figure 9

[0052] (A) Phenotypic analysis of Lactobacillus gasseri DSM 14869 (WT) and its mutant derivatives. Overnight cultures in MRS medium were vortexed and incubated at room temperature for 1 h. The mutants showed cell pellet and a very clear supernatant. In contrast, relatively homogeneous suspensions were observed for the WT and complement strains. (B) Quantitative analysis of the automated aggregation of WT and its mutant derivatives. Overnight cultures were washed and resuspended in PBS to 1.5 OD. 600 The suspension was allowed to stand undisturbed for 5 hours. The OD of the supernatant was measured. The percentage of autoaggregation was calculated using the following expression: Autoaggregation (%) = (1 - (OD) 600 5h / OD 600 0h))×100, where OD 600 5h represents the absorbance at a time point of 5 hours and the OD 600 0h represents absorbance at 0h. Autoaggregation capacity is shown as a percentage relative to the wild-type strain (set to 100%). Data are presented as mean ± SD of three independent experiments, *P < 0.05, ***P < 0.001. (C) Quantification of biofilm formation in WT and its mutant derivatives. Biofilm formation on polystyrene plates was assessed using crystal violet staining after 72 hours of incubation in MRS medium. Reads were taken at OD. 570 The absorbance values ​​are represented by the biofilm formation capacity. Data are expressed as mean ± SD of three independent experiments, ***P < 0.001.

[0053] Figure 10

[0054] Comparison of adhesion ability of wild-type, EPS mutant, and complement strain of Lactobacillus gasseri DSM 14869. Adhesion rates are shown as a percentage relative to wild-type (set to 100%). Each figure shows adhesion ability against (a) Caco-2 cells, (b) HeLa cells, and (c) vaginal epithelial cells. Data are expressed as mean ± SD of three independent experiments, **P < 0.01, ***P < 0.001.

[0055] Figure 11

[0056] mRNA expression of gene N506_1778 in WT, N506_1778 mutant (Δ1778), and N506_1778 complement (1778 complement) strains (A), and gene N506_1709 in WT, N506_1709 mutant (Δ1709), and N506_1709 complement (1709 complement) strains (B). mRNA expression of two genes in WT was used as a control. Data represent the mean ± SD of three independent experiments.

[0057] Figure 12

[0058] Comparison of the adhesion ability of *Lactobacillus gasseri* DSM 14869 wild-type, mutant strains Δ1778 and Δ1709. Adhesion rates are shown as a percentage relative to the wild-type (set to 100%). Each figure shows adhesion ability to (a) Caco-2 cells, (b) HeLa cells, and (c) vaginal epithelial cells. Data are expressed as mean ± SD from three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001.

[0059] Figure 13

[0060] (A) mRNA expression of N506_1709 in the overexpression strain *Lactococcus lactis* NZ9000 / pNZ8048-1709. *Lactococcus lactis* containing the empty pNZ8048 plasmid (NZ9000 / pNZ8048) was used as a control. (B) Adhesion ability of the overexpression strain NZ9000 / pNZ8048-1709 to vaginal epithelial cells. Adhesion rates are shown as percentages relative to NZ9000 / pNZ8048 (set as 100%). Data represent the mean ± SD of three independent experiments; ***P < 0.001.

[0061] Figure 14

[0062] Growth curves of Lactobacillus gasseri DSM 14869 wild type, N506_0400 mutant (ΔEPS), N506_1778 mutant (Δ1778), and N506_1709 mutant (Δ1709) strains in MRS medium.

[0063] Figure 15

[0064] (A) Plasmid profile of pINTZrec. This plasmid contains a multiple cloning site for integrating homologous fragments; two six sites and a β-recombinase (Rec), which specifically catalyzes recombination between the two six sites located on either side of the antibiotic resistance gene and the DNA to be integrated. Expression of the β-recombinase is controlled by the sakacin-inducible promoter. (B) Plasmid profile of pNZ8048. (C) Plasmid profile of pNZe-Rec. Detailed Implementation

[0065] In a first aspect, the present invention provides a circular DNA vector comprising:

[0066] (a) An optional marker gene sequence, wherein the marker gene sequence is operatively linked to a first promoter sequence.

[0067] (b) a multiple cloning site, wherein the multiple cloning site optionally includes a gene-targeting sequence.

[0068] (c) A sequence encoding a site-specific recombinase, wherein the sequence is operatively ligated to a second promoter sequence, wherein the second promoter is inducible.

[0069] (d) Copy the subsequence.

[0070] (e) Two target sites for the site-specific recombinase.

[0071] The vector comprises a first region flanked on each side by one of the target sites of the site-specific recombinase, and wherein said region comprises (a) and (b), provided that (c) and (d) are not within the first region. The circular DNA vector is preferably a plasmid.

[0072] The selectable marker can be any marker suitable for identifying and selecting host cells that express the marker. In one embodiment, the selectable marker is an antibiotic resistance gene, such as an antibiotic resistance gene selected from the group consisting of chloramphenicol resistance genes, spectinomycin resistance genes, tetracycline resistance genes, and erythromycin resistance genes.

[0073] The site-specific recombinase can be any site-specific recombinase suitable for genetic engineering. In one embodiment, the site-specific recombinase is a site-specific serine recombinase. In another embodiment, the site-specific recombinase is selected from the group consisting of β-recombinase, Cre-recombinase, FLP-recombinase, and PhiC31 integrase.

[0074] Target sites for site-specific recombinases are typically 30 to 200 nucleotides in length and usually consist of two motifs with partially inverted repeat symmetry. The recombinase binds to these motifs and is located flanking a central crossover sequence where recombination takes place. Examples of target sites include six (target site for β-recombinases), Lox (target site for Cre recombinases), and FRT (target site for FLP recombinases). Target sites for site-specific recombinases can be engineered to promote recombination. Single mutant sites, such as lox66 and lox71, can be used to generate double mutant sites as products of site-specific recombination. Double mutant sites are not substrates for site-specific recombinases and therefore prevent reversible site-specific recombination events.

[0075] In a preferred embodiment, the circular DNA vector contains two oriented target sites for the site-specific recombinase, such that the product of site-specific recombination between the two target sites for the site-specific recombinase is a first circular DNA product comprising (a) and (b) and a second circular DNA product comprising (c) and (d).

[0076] The circular DNA vector contains a replicon, preferably a prokaryotic replicon sequence, which allows the vector to replicate in a cloning host from which the vector can be harvested (e.g., *E. coli*). The replicon can be used for vector amplification and cloning purposes, such as cloning gene-targeting sequences. In one embodiment, the replicon sequence is a replicon sequence that allows the vector to replicate in *E. coli*, such as the origin of replication of pBR322. The circular DNA vector contains a replicon that allows replication in a host cell, subject to targeted insertion of the DNA vector (e.g., *Lactobacilli* or *Bifidobacteria*). In one embodiment, the circular DNA vector contains a replicon for replicating the vector in a cloning host and a further replicon for replicating the vector in a host cell, subject to targeted insertion of the DNA vector into the host cell genome. In another embodiment, the replicon sequence is a replicon sequence that allows the vector to replicate in *E. coli* and other prokaryotic host cells (e.g., host cells selected from the group consisting of *Lactobacilli* and *Bifidobacteria*). Therefore, in this embodiment, the replicon has a dual function: to replicate in the host cell, to amplify the vector, and to clone and replicate the vector in the host cell, subject to targeted insertion of the DNA vector into the host cell genome.

[0077] In one embodiment, the replicon sequence allows the vector to be selected from Lactobacillus gasseri (e.g., Lactobacillus gasseri DSM 14869), Lactobacillus rhamnosus (e.g., Lactobacillus rhamnosus DSM 14870), Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus vaginalis, Lactobacillus inertia, Lactobacillus reuteri, Lactobacillus casei, Lactobacillus bulgaricus, and Lactobacillus curvilinearus. A replicon sequence replicated in at least one host cell from the group consisting of *Lactobacillus curvatus*, *Lactobacillus delbrueckii*, and *Lactobacillus johnsonii*.

[0078] The resulting replicon sequence typically encodes the origin of replication (ori) and the replication initiation protein (Rep protein). In one embodiment, the replicon contains repA encoding the regulatory protein RepA, repB encoding the replication protein RepFIB, or RepC encoding the replication initiation protein.

[0079] The selectable marker gene sequence is operatively linked to a first promoter sequence. The first promoter guiding the expression of the selectable marker is preferably a promoter with constitutive activity and thus capable of expressing the marker in a host cell subject to targeted insertion of a DNA vector. Any suitable promoter and marker can be used.

[0080] To control the timing of site-specific recombination, site-specific recombinases are conditionally expressed, for example, by using any suitable inducible promoter. The choice of the promoter (second promoter) for expressing the recombinase depends on the host cell. In one embodiment, the second promoter sequence is an inducible prokaryotic promoter. In another embodiment, the second promoter sequence is selected from the group consisting of sakacin-inducible promoters, tetracycline-inducible promoters, D-xylose-inducible promoters, lactose-inducible promoters, IPTG-inducible promoters, nisin-inducible promoters, bile-inducible promoters, bacteriocin-inducible promoters, and synthetic inducible promoters.

[0081] The circular DNA vector contains a cloning site, preferably a multiple cloning site, which allows for the cloning of a gene-targeting sequence within the vector. In a preferred embodiment, the vector contains a gene-targeting sequence inserted into the multiple cloning site. The gene-targeting sequence contains a sequence with sufficiently high sequence identity to allow targeted integration of the vector into a corresponding target region in a host cell. Circular DNA vectors containing a gene-targeting sequence inserted into the cloning site are suitable for the methods of the present invention. The host cell is preferably a bacterial cell, more preferably a host cell consisting of *Lactobacillus* and *Bifidobacterium*. The vector can be introduced into the host cell using any suitable method, such as by transformation, electroporation, conjugation, or transduction.

[0082] The circular DNA vector preferably contains a replicon that allows the vector to replicate in the host cell, wherein the vector is to be inserted into the genome via homologous recombination. In a free state within the host cell, the vector can be rearranged by the expression of a site-specific recombinase, which (i) eliminates the vector's ability to replicate in the host cell and (ii) facilitates selection of homologous recombination events between the vector and chromosomal DNA. The products of the site-specific recombination events are a first circular DNA product and a second DNA product, wherein the first circular DNA product contains the selectable marker and a multiple cloning site in which a target sequence is inserted. The second circular DNA product contains a replicon and a sequence encoding a site-specific recombinase.

[0083] Selectable markers can be used to select for single-crossover genomic DNA integration (first homologous recombination event). Cells containing cell-free circular DNA vectors are restricted because the expression of site-specific recombinases eliminates the vector's ability to replicate in the host cell. Target-specific integration of the DNA vector (no longer circular) can be verified, for example, by PCR. The host can reproduce to allow for further (second) homologous recombination events, the product of which is the excision of the selectable marker. Hosts that have undergone two homologous recombination events can be identified by anti-selection through the loss of the selectable marker, such as sensitivity to antibiotics using antibiotic resistance markers. Proper double-crossover should be preferentially identified using methods such as PCR and / or sequencing.

[0084] A second aspect of the invention provides a method for introducing recombination between a circular DNA vector and a target region of a host cell genome, the method comprising the steps of:

[0085] (i) Introducing a circular DNA vector containing a gene-targeting sequence according to the invention into a host cell, wherein the target sequence comprises flanking sequences containing at least about 200 consecutive nucleotides having at least 80% sequence identity with a corresponding region of the target region of the host cell genome.

[0086] (ii) Inducing the expression of a site-specific recombinase encoded by the circular DNA vector, and allowing site-specific recombination between target sites of the site-specific recombinase to produce a first circular DNA product comprising (a) and (b) and a second circular DNA product comprising (c) and (d).

[0087] (iii) Selecting a host cell, wherein the first circular DNA product comprising (a) and (b) is integrated into the target region of the genome via a first single crossover homologous recombination event between the flanking sequence of the target sequence and the target region of the host cell genome, and

[0088] (iv) Select a host cell in which (a) has been excised from the genome of the host cell obtained in (iii) by a second homologous recombination event between the flanking sequence of the target sequence and the target region of the genome.

[0089] The circular DNA vector of this invention can be used to introduce specific mutations in a target region of a host cell. The target region can be a coding region, such as a gene encoding a protein. The mutation can be a point mutation that alters or disrupts the coding sequence. The coding sequence can also be disrupted by mutations that introduce deletions or insertions into the sequence. Alternatively, the mutation can alter (decrease or increase) or restore the activity of the encoded protein.

[0090] Therefore, a third aspect of the present invention provides a method for generating host cells with mutations in target genes of the host cell genome, the method comprising the steps of:

[0091] (i) Introducing a circular DNA vector containing a gene-targeting sequence according to the invention into a host cell, wherein the target sequence comprises flanking sequences containing at least about 200 consecutive nucleotides having at least 80% sequence identity with a corresponding region of a target gene in the host cell genome.

[0092] (ii) Inducing the expression of a site-specific recombinase encoded by the circular DNA vector, and allowing site-specific recombination between target sites of the site-specific recombinase to produce a first circular DNA product comprising (a) and (b) and a second circular DNA product comprising (c) and (d).

[0093] (iii) Selecting a host cell, wherein the first circular DNA product comprising (a) and (b) is integrated into the target region of the genome via a first single crossover homologous recombination event between the flanking sequence of the target sequence and the target region of the host cell genome, and (iv) Selecting a host cell, wherein (a) has been excised from the genome of the host cell obtained in (iii) via a second homologous recombination event between the flanking sequence of the target sequence and the target region of the genome, and wherein the host cell contains a mutation in the target region.

[0094] The method can be used, for example, to evaluate the function of a specific gene. For instance, it can evaluate whether a specific gene has a function in, for example, bacterial adhesion, autodistribution, and / or biofilm formation.

[0095] In a related aspect, the present invention provides a method for generating host cells with loss of function in target genes of the host cell genome, the method comprising the steps of:

[0096] (i) Introducing a circular DNA vector containing a gene-targeting sequence according to the invention into a host cell, wherein the target sequence comprises flanking sequences containing at least about 200 consecutive nucleotides having at least 80% sequence identity with a corresponding region of a target gene in the host cell genome.

[0097] (ii) Inducing the expression of a site-specific recombinase encoded by the circular DNA vector, and allowing site-specific recombination between target sites of the site-specific recombinase to produce a first circular DNA product comprising (a) and (b) and a second circular DNA product comprising (c) and (d).

[0098] (iii) Selecting a host cell, wherein the first circular DNA product comprising (a) and (b) is integrated into the target region of the genome via a first single crossover homologous recombination event between the flanking sequence of the target sequence and the target region of the host cell genome, and (iv) Selecting a host cell, wherein (a) has been excised from the genome of the host cell obtained in (iii) via a second homologous recombination event between the flanking sequence of the target sequence and the target region of the genome, and wherein the host cell contains the target gene and is rendered nonfunctional.

[0099] As described above, this method can be used, for example, to assess the function of a specific gene. Functional analysis can be used to address the potential impact of targeted engineering leading to loss of gene function in the target region on host cell phenotypic traits (compared to wild-type host cells). Phenotypic traits include, but are not limited to, bacterial adhesion (e.g., to mammalian tissues), autoaggregation, and / or biofilm formation.

[0100] Another aspect of the present invention provides a method for generating host cells with functional gains in target genes of the host cell genome, the method comprising the steps of:

[0101] (i) Introducing a circular DNA vector containing a gene-targeting sequence according to the invention into a host cell, wherein the target sequence comprises flanking sequences containing at least about 200 consecutive nucleotides having at least 80% sequence identity with the target gene of the host cell genome.

[0102] (ii) Inducing the expression of a site-specific recombinase encoded by the circular DNA vector, and allowing site-specific recombination between target sites of the site-specific recombinase to produce a first circular DNA product comprising (a) and (b) and a second circular DNA product comprising (c) and (d).

[0103] (iii) Selecting a host cell, wherein the first circular DNA product comprising (a) and (b) is integrated into the target region of the genome via a first single crossover homologous recombination event between the flanking sequence of the target sequence and the target region of the host cell genome, and (iv) Selecting a host cell, wherein (a) has been excised from the genome of the host cell obtained in (iii) via a second homologous recombination event between the flanking sequence of the target sequence and the target region of the genome, and wherein the host cell contains functional gain of the target gene.

[0104] This method can be used, for example, to assess the reversal and recovery of lost phenotypic features in host cells from previously lost gene function, such as bacterial adhesion (e.g., in mammalian tissues), autoaggregation, and / or biofilm formation.

[0105] The target gene can encode any gene product. Target genes typically encode proteins. In one embodiment, the target gene encodes a cell surface protein. In one embodiment, the target gene encodes a protein involved in bacterial adhesion, autoaggregation, and / or biofilm formation. In another embodiment, the cell surface protein is a sorting enzyme-dependent protein (SDP) or an S-layer protein, or encodes a protein involved in the biosynthesis of cell surface molecules. In one embodiment, the cell surface molecule is an extracellular polysaccharide (EPS). In one particular implementation, the target gene is a gene selected from the group consisting of N506_1709, N506_1778, N506_0396, N506_0397, N506_0398, N506_0399, N506_0400, N506_0401, N506_0402, N506_0403, N506_0404, N506_0405, N506_0406, N506_0407, N506_0408, N506_0409, N506_0410, and N506_0411.

[0106] In one embodiment, the host cell is *Lactobacillus gasseri* (e.g., *Lactobacillus gasseri* DSM14869) and the target gene is selected from the group consisting of N506_1709, N506_1778, N506_0396, N506_0397, N506_0398, N506_0399, N506_0400, N506_0401, N506_0402, N506_0403, N506_0404, N506_0405, N506_0406, N506_0407, N506_0408, N506_0409, N506_0410, and N506_0411.

[0107] The circular DNA vector of the present invention can be used to prepare host cell recombinant expression of recombinant gene products (e.g., recombinant proteins). In this respect, the gene targeting sequence typically includes a sequence having sufficient sequence identity with the target region to allow for target region-specific integration; and a sequence encoding the recombinant gene product (e.g., a polypeptide).

[0108] Therefore, in a further aspect, the present invention provides a method for preparing host cells expressing recombinant peptides, the method comprising the steps of:

[0109] (i) Introducing a circular DNA vector comprising a gene-targeting sequence according to the invention into a host cell, wherein the target sequence comprises flanking sequences containing at least about 200 consecutive nucleotides having at least 80% sequence identity with a corresponding region of the target region of the host cell genome, and wherein the gene-targeting sequence encodes a recombinant polypeptide.

[0110] (ii) Inducing the expression of a site-specific recombinase encoded by the circular DNA vector and allowing site-specific recombination between target sites of the site-specific recombinase to produce a first circular DNA product comprising (a) and (b) and a second circular DNA product comprising (c) and (d).

[0111] (iii) Selecting a host cell, wherein the first circular DNA product comprising (a) and (b) is integrated into the target region of the genome via a first single crossover homologous recombination event between the flanking sequence of the target sequence and the target region of the host cell genome, and

[0112] (iv) Select a host cell in which (a) has been excised from the genome of the host cell obtained in (iii) by a second homologous recombination event between the flanking sequence of the target sequence and the target region of the genome.

[0113] In one embodiment, the recombinant polypeptide is selected from the group consisting of antibodies (such as monoclonal antibodies, humanized monoclonal antibodies, chimeric antibodies, single-domain antibodies, camel antibodies), enzymes, cytokines, hormones, and blood clotting proteins. The host cells obtained by this method can be used as production cells for the recombinant product.

[0114] The gene-targeting sequence includes a sequence in a flanking region that has a sufficiently high identity with the target region sequence to allow / promote homologous recombination. In one embodiment, the sequence in the flanking region (flanking sequence) comprises 200 to 1500 consecutive nucleosides, for example, at least about 300 consecutive nucleosides, such as at least about 400 consecutive nucleosides, such as at least about 500 consecutive nucleosides, such as at least about 600 consecutive nucleosides, such as at least about 700 consecutive nucleosides, such as at least about 800 consecutive nucleosides, such as at least about 900 consecutive nucleosides, such as at least about 1000 consecutive nucleosides, such as at least about 1100 consecutive nucleosides, such as at least about 1200 consecutive nucleosides, such as at least about 1300 consecutive nucleosides, such as at least about 1400 consecutive nucleosides, such as at least about 1500 consecutive nucleosides.

[0115] In another embodiment, the flanking sequence has at least 85% sequence identity with the corresponding region of the host cell genome target gene, for example, at least 95% sequence identity with the corresponding region of the host cell genome target gene, for example, at least 97% sequence identity with the corresponding region of the host cell genome target gene, for example, at least 98% sequence identity with the corresponding region of the host cell genome target gene, for example, at least 99% sequence identity with the corresponding region of the host cell genome target gene, for example, at least 100% sequence identity with the corresponding region of the host cell genome target gene.

[0116] In one embodiment, the selection under (iii) uses an optional marker gene sequence (a) of a circular DNA vector (selected for host cells with single cross-integration of the DNA vector). Host cells containing single cross-integration of the DNA vector can be identified / verified using PCR and / or DNA sequencing. Therefore, in one embodiment, the selection under (iii) uses PCR and / or DNA sequencing, such as sequencing the sequence connections between the target sequence and the target region.

[0117] The host can reproduce to allow for further (second) homologous recombination events, the product of which is the excision of a selectable marker. Hosts that have undergone two homologous recombination events can be identified by antiselection through the loss of the selectable marker, for example, leading to sensitivity to antibiotics using antibiotic resistance markers. The correct double-cross event should preferably be identified using methods such as PCR and / or DNA sequencing. In a preferred embodiment, host cells that have undergone both the first and second homologous recombination events are selected and / or verified using PCR or DNA sequencing.

[0118] The product of the method of the present invention (the product of (iv)) is usually a host cell, which includes the deletion of the target region, the partial deletion of the target region, the insertion of the target region sequence, the point mutation of the target region, or the sequence substitution of the target region.

[0119] The host cell is preferably a prokaryote, and more preferably a bacterium. In a preferred embodiment, the host cell is *Lactobacillus* or *Bifidobacterium*. In another embodiment, the host cell is selected from the group consisting of *Lactobacillus gasseri* (e.g., *Lactobacillus gasseri* DSM 14869), *Lactobacillus rhamnosus* (e.g., *Lactobacillus rhamnosus* DSM 14870), *Lactobacillus paracasei*, *Lactobacillus plantarum*, *Lactobacillus acidophilus*, *Lactobacillus fermentum*, *Lactobacillus curvilinearus*, *Lactobacillus janniae*, *Lactobacillus vaginalis*, *Lactobacillus indolent*, *Lactobacillus reuteri*, *Lactobacillus casei*, *Lactobacillus bulgaricus*, *Lactobacillus curvilinearus*, *Lactobacillus delbrueckii*, and *Lactobacillus johnsonii*. In a preferred embodiment, the host cell is *Lactobacillus gasseri* (e.g., *Lactobacillus gasseri* DSM 14869).

[0120] In a further aspect, the present invention provides recombinant host cells obtainable by the method of the present invention. In one embodiment, the host cell is Lactobacillus or Bifidobacterium. In another embodiment, the host cell is Lactobacillus gasseri (such as Lactobacillus gasseri DSM 14869). In another embodiment, the host cell is *Lactobacillus gasseri* (e.g., *Lactobacillus gasseri* DSM14869), which is engineered to express genes selected from the group consisting of N506_1709, N506_1778, N506_0396, N506_0397, N506_0398, N506_0399, N506_0400, N506_0401, N506_0402, N506_0403, N506_0404, N506_0405, N506_0406, N506_0407, N506_0408, N506_0409, N506_0410, and N506_0411. In a preferred embodiment, the Lactobacillus gasseri (e.g., Lactobacillus gasseri DSM 14869) is engineered to express N506_1709 and / or N506_1778.

[0121] In one embodiment, the coding region of the gene is operatively linked to a recombinant promoter, preferably a recombinant constitutive promoter.

[0122] One aspect of the invention provides the use of the circular DNA vector of the invention for introducing a gene sequence into the genome of a host cell. In one embodiment, the circular DNA vector is used to increase tissue adhesion of host cells, such as to increase the tissue adhesion of bacterial host cells to vaginal tissue (preferably human vaginal tissue). The host cell is preferably a lactobacillus or a bifidobacterium. In one embodiment, the host cell is selected from the group consisting of *Lactobacillus gasseri* (e.g., *Lactobacillus gasseri* DSM14869), *Lactobacillus rhamnosus* (e.g., *Lactobacillus rhamnosus* DSM 14870), *Lactobacillus paracasei*, *Lactobacillus plantarum*, *Lactobacillus acidophilus*, *Lactobacillus fermentum*, *Lactobacillus curvilinearus*, *Lactobacillus janniae*, *Lactobacillus vaginalis*, *Lactobacillus indolentus*, *Lactobacillus reuteri*, *Lactobacillus casei*, *Lactobacillus bulgaricus*, *Lactobacillus curvilinearus*, *Lactobacillus delbrueckii*, and *Lactobacillus johnsonii*.

[0123] In one implementation, the circular DNA vector is used to introduce a deletion, partial deletion, insertion, point mutation, or substitution of a target region.

[0124] In one embodiment, the circular DNA vector is used to target genes selected from the group consisting of N506_0396, N506_0397, N506_0398, N506_0399, N506_0400, N506_0401, N506_0402, N506_0403, N506_0404, N506_0405, N506_0406, N506_0407, N506_0408, N506_0409, N506_0410, and N506_0411 in host cells of Lactobacillus gasseri (e.g., Lactobacillus gasseri DSM 14869).

[0125] In another embodiment, the circular DNA vector is used to introduce and express the gene sequence in the host cell. In yet another embodiment, the gene sequence blocks the expression of an endogenous host gene. In a further embodiment, the gene sequence replaces a corresponding endogenous host gene.

[0126] In one embodiment, the circular DNA vector is used to increase tissue adhesion of bacterial host cells, such as increasing the tissue adhesion of bacterial host cells (e.g., Lactobacillus or Bifidobacterium) to vaginal tissue (preferably human vaginal tissue). In one embodiment, the host cells are selected from the group consisting of Lactobacillus gasseri (e.g., Lactobacillus gasseri DSM 14869), Lactobacillus rhamnosus (e.g., Lactobacillus rhamnosus DSM 14870), Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus curvifolius, Lactobacillus janniae, Lactobacillus vaginalis, Lactobacillus indolentus, Lactobacillus reuteri, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus curvifolius, Lactobacillus delbrueckii, and Lactobacillus johnsonii.

[0127] Example

[0128] Example 1

[0129] A novel vector is provided that can be conditionally disrupted to facilitate the selection of homologous recombination events between the vector and chromosomal DNA. Essentially, the replicon and the antibiotic resistance gene are separated by two oriented six fragments. Upon addition of an inducer and expression of a site-specific recombinase (β), the vector recombines and loses its function due to the excision of the replicon. Figure 1 a). This vector, called induced plasmid self-destruction (IPSD), can be used to assist bacterial recombination engineering, including gene deletion, insertion, and substitution. Figure 1 b). As a proof of concept, the IPSD plasmid pINTZrec was constructed based on the β-six recombination system. Figure 2 ), of which the β-recombinase gene is influenced by the sakacin-inducible promoter P orfXThe control of chloramphenicol resistance was observed. When the plasmid pINTZrec carrying the chloramphenicol resistance gene was introduced into the vaginal probiotic strain *Lactobacillus gasseri* DSM14869, the pINTZrec transformants showed significant sensitivity to Sakacin P (SppIP) induction. Compared with the non-induced control, the viability of SppIP-induced pINTZrec transformants on MRS agar plates supplemented with chloramphenicol was reduced by three to four orders of magnitude. Figure 1 c) indicates that the plasmid pINTZrec is destroyed after β-recombinase expression. This observation has also been found in other lactic acid bacteria species (Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus plantarum). Figure 4 ).

[0130] Previous work by the inventors has shown that the transformation efficiency of *Lactobacillus gasseri* DSM 14869 is very low, possibly due to the thickness of EPS covering the cell surface or the presence of two resident plasmids, making it impossible to generate mutations in this strain using existing methods (Marcotte, H. et al. (2017) and unpublished data). To demonstrate IPSD-assisted recombination engineering in *Lactobacillus gasseri* DSM 14869, we targeted upp, a non-essential gene encoding uracil phosphoribosyltransferases (UPRTases), which are commonly used as anti-selection markers. A recombinant plasmid pINTZrec-Δupp containing upstream and downstream homologous regions of the upp gene was constructed and introduced into *Lactobacillus gasseri* DSM 14869. Transformants were induced by SppIP, and single crossover integration events were selected using colony PCR. Figure 3 a, Figure 5a). Of the 28 randomly selected colonies, 10 (36%) showed no plasmid pINTZrec-Δupp, indicating that Cm r The expression cassette integrated into the chromosome of *Lactobacillus gasseri* DSM 14869 via a single crossover event. These results were further confirmed by PCR of DNA extracted from isolated clones, with six clones showing correct integration (Fig. 5b). Following growth of single crossover clones in the absence of antibiotics, Cm could be selected via reverse selection. s Double-cross UPP mutants can be easily selected by colony selection or by selecting colonies resistant to 5-fluorouracil (5-FU). Figure 3 b, Figure 5c). Due to the elimination of 5-FU and conversion to cytotoxic 5-fluorodeoxyuridine monophosphate (5-FdUMP), the *Lactobacillus gasseri* DSM14869upp mutant showed resistance to 5-FU (100 μg / ml) compared to the parent strain. Figure 3c). We also used this method to generate mutants of several cell surface characteristic-related genes and the integrated expression of a broad and effective HIV-1-neutralizing antibody in Lactobacillus gasseri DSM 14869 (unpublished data). These results demonstrate that IPSD plasmids can be effectively used for genome engineering in Lactobacillus.

[0131] The main advantage of IPSD-assisted bacterial recombination engineering is that it is independent of transformation (or conjugation) efficiency. However, it requires two prerequisites: 1) a functional replicon that allows the plasmid to replicate in the host bacteria; and 2) a tightly controlled expression element to drive the expression of the recombinase gene. Due to strong background expression (Lactobacillus sakei NC03) Figure 6 a) or low-inducible expression (Lactobacillus rhamnosus GG) Figure 6 (b) The inducible promoter used in this study was ineffective in some strains. Therefore, it is desirable to develop a universal, tightly controlled lactobacillus expression element, such as a tetracycline regulatory system, for Lactobacillus.

[0132] In summary, the inventors have demonstrated that IPSD plasmids can be used for genome engineering in Lactobacillus and have the potential to be extended to other bacterial species. The IPSD strategy can be used in a range of applications in the food and pharmaceutical industries, such as the identification of probiotic genes, metabolic engineering, and therapies, thereby opening new avenues for the engineering design of biotherapeutic agents with enhanced health-promoting properties.

[0133] method

[0134] Bacterial strains, plasmids, and growth conditions

[0135] Table 1 lists the bacterial strains and plasmids used in this study. Lactobacillus strains were typically cultured at 37°C in deMan Rogosa Sharpe (MRS) medium (Difco, BD BioSciences). For upp-based double crossover event selection, Lactobacillus strains were grown on semi-limited media (SDM) agar plates (Kimmel et al.). Escherichia coli strains were cultured in Luria-Bertani broth at 37°C with shaking at 200 rpm or on LB agar plates. Antibiotics were supplemented as needed at the following concentrations: Lactobacillus strains and Escherichia coli VE7108 strain, 10 μg / ml chloramphenicol.

[0136] plasmid construction

[0137] Table 2 lists the primers used in this study. To construct the IPSD vector pINTZrec for Lactobacillus recombinant engineering, two six DNA fragments were amplified from the site-specific integration vector pEM76 using primer pairs SIX-F1&R1 and SIX-F2&R2, respectively, and generated at Cm. r The expression cassette was inserted flanking the plasmid pNZ8048. Insertion orientation was confirmed by sequencing. The plasmid pNZmcs-SIX was generated by inserting an adapter between PstI and BglII to introduce a multiple cloning site in pNZ8048-SIX. The β-recombinase gene was amplified from plasmid pEM94 and inserted into plasmid pVPL3017 at the sakacin-inducible promoter P. orfX Downstream, pVPL3017-rec is produced. Subsequently, P is digested using SalI and HindIII. orfX The -rec expression cassette was inserted into a similarly digested pNZmcs-SIX plasmid to obtain the final plasmid pINTZrec.

[0138] To construct a plasmid for the deletion of the upp gene in *Lactobacillus gasseri* DSM 14869, two 1090 bp DNA fragments upstream and downstream of the upp gene were amplified from the genomic DNA of *Lactobacillus gasseri* DSM14869 using primer pairs upp-up-F / upp-up-R and upp-down-F / upp-down-R, respectively. The upstream DNA fragment was inserted into the pMD19-T simple vector via TA cloning to generate pMD19-upp-up, and the downstream DNA fragment was then inserted between SacI and SphI in pMD19-upp-up to generate pMD19-Δupp. Δupp was digested with ApaI and SphI and then inserted into a similarly digested pINTZrec vector to generate pINTZrec-Δupp.

[0139] Transformation

[0140] According to De Keersmaecker et al, plasmids pINTZrec and pINTZrec-Δupp were electroporated into *Lactobacillus gasseri* DSM 14869 and other *Lactobacillus* strains. Plasmid pNZ8048 was used as a control. Transformations were confirmed by colony PCR, and then DNA extracted from pure cultures was subjected to PCR.

[0141] Restructuring Project

[0142] Single colonies of *Lactobacillus gasseri* DSM 14869 with plasmid pINTZrec or a derivative thereof were grown overnight in MRS broth containing 10 μg / ml chloramphenicol. The cultures were then inoculated into (1%, v / v) antibiotic-free MRS broth and grown at 37°C until OD500. 600nm Reach approximately 0.30, then supplement with 100 ng / ml sakacin P (SppIP) (Genscript). Allow the culture to grow overnight, and inoculate serial dilutions onto MRS agar supplemented with 10 μg / ml chloramphenicol and 100 ng / ml SppIP. Single crossover events are detected by colony PCR, followed by PCR of DNA extracted from pure cultures. For double crossover selection, single crossover clones are grown overnight in antibiotic-free MRS broth, then serial dilutions are spread onto MRS agar or SDM agar supplemented with 100 μg / ml 5-fluorouracil (5-FU). Colonies from MRS agar are replicated onto MRS agar containing 10 μg / ml chloramphenicol, selecting Cm... s The colonies were analyzed and the extracted DNA was detected by PCR.

[0143] All mutants generated in this study were confirmed by PCR and sequencing.

[0144] Table 1. Strains and plasmids.

[0145]

[0146]

[0147] a Km r Kanamycin resistance; Cm r ; Chloramphenicol resistance; Sm r Spectinomycin resistance.

[0148] Table 2. Oligonucleotides used in this study

[0149]

[0150]

[0151] a The restriction sites are underlined.

[0152] Example 2

[0153] Lactobacilli play a vital role in maintaining a healthy vaginal microbiota, and several selected species are widely used as probiotics. Previously, vaginal isolates of *Lactobacillus gasseri* DSM 14869 and *Lactobacillus rhamnosus* DSM 14870 were selected for probiotic development. Capsules have been shown to have therapeutic effects on women with bacterial vaginosis (BV). However, the molecular basis and mechanisms involved in their probiotic activity are largely unknown. In this study, by constructing specific knockout mutants, we identified three cell surface molecules that promote adhesion to vaginal epithelial cells in *Lactobacillus gasseri* DSM 14869, including extracellular polysaccharides (EPS), a protein containing a MucBP-like domain (N506_1778), and a putative new adhesin with Rib / α-like domain repeats (N506_1709). Compared to wild type, the EPS knockout mutant increased adhesion to Caco-2 and HeLa cells by 20-fold and 14-fold, respectively, while decreasing adhesion to vaginal cells by 30%, suggesting that EPS may mediate tissue tropism of vaginal cells. Significantly reduced adhesion to Caco-2, HeLa, and vaginal cells was observed in the N506_1778 knockout mutant. Compared to WT, the N506_1709 mutant showed no significant difference in adhesion to Caco-2 and HeLa cells; however, adhesion to vaginal cells was significantly reduced (42%), suggesting that N506_1709 may mediate specific binding to layered squamous epithelial cells. This hypothetical new adhesin has been annotated as Lactobacillus vaginal epithelial cell adhesin (LVEA). Therefore, the inventors have for the first time discovered the important roles of EPS and the novel adhesin LVEA in the adhesion ability of the vaginal probiotic Lactobacillus strain.

[0154] Lactobacilli are known to contribute to maintaining a healthy vaginal microbiota, and some have been selected as probiotics for the prevention or treatment of urogenital diseases such as bacterial vaginosis. However, the molecular mechanisms underlying these health-promoting effects are poorly understood. Here, we functionally identify three cell surface factors in Lactobacillus gasseri strains that may be involved in adhesion to vaginal epithelial cells, including extracellular polysaccharides (EPS) and two sorting enzyme-dependent proteins (N506_1778 and N506_1709). For the first time, we have demonstrated tissue-specific adhesion of EPS to vaginal cells, and that N506_1709 may be a novel adhesin that specifically mediates bacterial binding to stratified squamous epithelial cells. These results provide important new information about the molecular mechanisms of vaginal lactobacillus adhesion.

[0155] introduction

[0156] The vaginal microbiota of healthy women is typically dominated by lactobacilli, with the most common species being *Lactobacillus curvularia*, *Lactobacillus gasseri*, *Lactobacillus janniae*, *Lactobacillus vaginalis*, and *Lactobacillus indolentus* (Pendharkar et al. 2013; Ravel et al. 2011; Vasquez et al. 2002). These bacteria maintain a normal vaginal microbiota by adhering to vaginal epithelial cells (VECs) and preventing the growth of pathogenic organisms (Ronnqvist et al. 2006). Once the balance of the local microbiota is disrupted, women become susceptible to urogenital infections such as bacterial vaginosis (BV) (Danielsson et al. 2011). Providing selected lactobacilli may be a reasonable therapeutic strategy for restoring a healthy microbiota and preventing infection (Bolton et al. 2008; Reid et al. 2009). To meet this challenge, two strains isolated from vaginal epithelial cells of healthy women (Lactobacillus gasseri DSM14869 and Lactobacillus rhamnosus DSM 14870, 10 strains of each strain) have previously been developed. 8 The business comprised of CFU) Vaginal capsules, and Vaginal administration of the capsules eliminated BV symptoms in 90% of patients (Stray-Pedersen et al. 2003, unpublished data). Vaginal use... Capsule supplementation therapy can also effectively improve the efficacy of antibiotic treatment for BV (Larsson et al. 2008; 2011; Pendharkaret et al. 2015). Despite extensive clinical data showing... While the strains offer health benefits to humans, our understanding of the cell surface factors or molecular mechanisms underlying their probiotic activity remains limited.

[0157] Studies of gastrointestinal lactobacilli have shown that the health-promoting effects of probiotics may be related to their ability to adhere to intestinal epithelial cells and / or mucus, as this can promote colonization, pathogen rejection, and host-probiotic interactions (Lebeer et al. 2008; 2010). Therefore, high adhesion to the intestinal surface is considered a key differentiating characteristic for selecting probiotic strains. Bacterial adhesion to the host epithelial surface is typically mediated by cell surface components, including sorting enzyme-dependent proteins (SDPs) and other cell surface molecules such as extracellular polysaccharides (EPS), lipoteichoic acid, and S-layer proteins (Lebeer et al. 2008), and extracellular appendages such as pili, cilia, and flagella (Juge, 2012).

[0158] EPS significantly promotes Lactobacillus-host interactions, particularly with the intestinal mucosa and epithelial cells, thereby contributing to strain-specific probiotic properties (Lebeer et al., 2008). Bacterial polysaccharides vary in sugar composition, branching location, and modifications, leading to extensive diversity in surface structures (Ruas-Madiedo et al., 2002). EPS has been reported to participate in probiotic strain aggregation, biofilm formation, adhesion properties, and immune regulation (Lebeer et al., 2009; Dertli et al., 2015; Lee et al., 2016). et al. 2016).

[0159] SDPs are an important group of cell surface proteins in Gram-positive bacteria, exhibiting the best performance in Lactobacillus and demonstrating a key role in bacterial adhesion (Boekhorst et al. 2005). These SDPs share a common structure, including a secretion-promoting YSIRK signal peptide (Bae & Schneewind, 2003), a C-terminal LPxTG anchoring motif, followed by a transmembrane helix and a positively charged tail (Lebeer et al. 2008; Jensen et al. 2014). After translocation to the plasma membrane, the surface protein precursors are cleaved by sorting enzyme A and covalently anchored to the cell wall (Marraffini et al. 2006). Different SDPs have been identified in lactobacilli, including SpaC (mucus-binding fimbriae) in *Lactobacillus rhamnosus* GG (Kankainen et al. 2009), Lactobacillus epithelial adhesion (LEA) in *Lactobacillus curvature* ST1 (Edelman et al. 2012), CmbA (mucus-binding protein A) in *Lactobacillus reuteri* ATCC PTA6475 (Jensen et al. 2014), and mannose-specific adhesin (MSI) in *Lactobacillus plantarum* CMPG5300 (Malik et al. 2016). However, to date, few SDPs have been identified in human vaginal lactobacilli.

[0160] In previous research, the inventors had discovered The strains exhibited highly efficient adhesion to vaginal epithelial cells. We also performed complete genome sequencing and characterization of both strains, identifying several genes potentially related to bacterial probiotic activity (Marcotte et al. 2017). Specifically, a thick (40 nm) EPS layer and the production of a novel adhesin containing three rib / α-like repeats were found in *Lactobacillus gasseri* DSM 14869 (Marcotte et al. 2017). In this study, the inventors aimed to characterize the surface molecules of *Lactobacillus gasseri* DSM 14869 that mediate adhesion to human vaginal mucosa, including EPS, a protein with a mucus-binding-like domain (N506_1778), and a novel adhesin with a rib / α-like repeat domain (N506_1709). The results indicate that the genes encoding EPS, N506_1778, and N506_1709 contribute to the ability of *Lactobacillus gasseri* DSM 14869 to adhere to vaginal epithelial cells in vitro.

[0161] result

[0162] Identification of the putative EPS gene cluster in Lactobacillus gasseri DSM 14869. The genome of Lactobacillus gasseri DSM14869 (Marcotteet al. 2017) contains a putative EPS cluster, which consists of 16 genes (N506_0396 to N506_0411) that share high similarity with Lactobacillus gasseri ATCC 33323 (Azcarate-Perilet al. 2008). Figure 7 A) Composition. These genes are expected to be involved in EPS biosynthesis ( Figure 7 A) This includes those encoding glycosyltransferases and proteins involved in polymerization, export, and chain length determination (Lebeer et al. 2009). Based on BlastP analysis, the N506_0400 gene encodes a putative initiating glycosyltransferase protein that shares 91% amino acid homology with the initiating glycosyltransferase epsE in *Lactobacillus johnsonii* FI9785 (Horn et al. 2013). Initiating glycosyltransferases have been shown to be essential control points in EPS biosynthesis (Horn et al. 2013), and we therefore hypothesize that the deletion of the putative initiating glycosyltransferase gene (N506_0400) would affect EPS production in *Lactobacillus gasseri* DSM 14869.

[0163] The deletion of the N506_0400 gene affected the overall level of EPS. TEM images clearly showed that the N506_0400 gene deletion mutant strain (ΔEPS) produced a significantly (p<0.001) smaller EPS layer around the cell surface compared to the wild-type (WT) strain. Figure 8Meanwhile, reintroducing the functional N506_0400 gene into the mutant strain completely restored the thickness of the EPS layer to the WT level.

[0164] The deletion of the N506_0400 gene leads to increased autoaggregation and biofilm formation. When the N506_0400 mutant strain *Lactobacillus gasseri* DSM 14869-ΔN506_0400 (ΔEPS) was grown in liquid medium, no significant difference in growth rate was observed compared to WT *Lactobacillus gasseri* DSM14869. Figure 14 However, the mutant showed cell precipitation and a very clear supernatant, while the WT strain showed a relatively homogeneous suspension. Figure 9A The complement strain restored the phenotype to WT levels, showing a homogeneous suspension. Figure 9A The mutant strain *Lactobacillus gasseri* DSM 14869-ΔN506_0400 also showed a significant increase in autoaggregation ability (p<0.001), increasing to 216% of the WT. Autoaggregation in complement strains recovered to 136%. Figure 9B Furthermore, biofilm formation in the mutant strain was increased 15-fold compared to that in the WT strain, as assessed by microtitration biofilm assay (p<0.001). Figure 9C Complement partially restored biofilm formation, increasing it by 8-fold compared to WT. Figure 9C ).

[0165] The effect of EPS on the adhesion of Lactobacillus gasseri DSM 14869 to different epithelial cells. Previous studies have shown that EPS from intestinal lactobacilli are involved in adhesion to intestinal epithelial cells (Lebeer et al. 2009; Lee et al. 2016; (et al. 2016). In this study, we investigated the role of EPS in the adhesion ability of the vaginal strain *Lactobacillus gasseri* DSM 14869. For example, in... Figure 10A The observed adhesion of the EPS mutant *Lactobacillus gasseri* DSM14869-ΔN506_0400 to the Caco-2 colon cancer cell line was significantly increased by 20-fold compared to the WT strain (p<0.001). Furthermore, the EPS mutant also showed a significant increase of approximately 14-fold in adhesion to the HeLa cervical cancer cell line (p<0.001). Figure 10B Mutant strains supplemented with the pNZe-N506_0400 gene showed that their adhesion to Caco-2 and HeLa cells was restored to near-WT levels. Figure 10A (B) We further investigated whether ESP is involved in vaginal epithelial cell adhesion. Interestingly, the EPS mutant strain showed a slight decrease in vaginal epithelial cell adhesion (approximately 30%, p < 0.01). Figure 10C The complement strain (14869-ΔN506_0400 / pNZe-N506_0400) showed partial recovery of adhesion. Figure 10C This suggests that EPS in Lactobacillus gasseri DSM 14869 can promote heterotaxis towards vaginal epithelial cells.

[0166] Sequence analysis of putative adhesion proteins in Lactobacillus gasseri DSM 14869. Surface proteins are crucial molecules involved in bacterial adhesion to mucus and epithelial cells. In the genome of Lactobacillus gasseri DSM 14869, 22 predicted ORFs with putative adhesion-associated domains have previously been identified (Marcotte et al. 2017). However, only two ORFs (N506_1778 and N506_1709) encode the YSIRK-type signal peptide (PF04650) and LP... X The TG anchoring motif (PF00746) leads to protein secretion and covalent anchoring to the cell wall. Figure 7 (B, C). Therefore, these two proteins are classified as SDPs and both may be involved in the adhesion of Lactobacillus gasseri DSM 14869 to epithelial cells.

[0167] The N506_1778 gene in *Lactobacillus gasseri* DSM 14869 is 5.055 kb long and encodes a protein with 1684 amino acid residues and a predicted molecular weight of 186.7 kDa. It has a YSIRK-type signal peptide at the N-terminus and contains LPQTG (LP) at the C-terminus. X TG-like cell wall anchor motifs, which belong to Gram-positive LPs X The TG-anchored superfamily. The N506_1778 protein also contains two distinct repeats (amino acids 986-1092 and 1384-1490) showing 66% amino acid identity. Figure 7B). The two repeats showed low homology with the MucBP (mucin-binding protein) (PF06458) domain, 35%–39% aa identity with the Mub-RV repeat of the mucin-binding protein (MUB) from L. reuteri ATCC 53608 (Etzold et al. 2014), 31–37% aa identity with the Mub1 repeat of the MUB from L. reuteri 1063 (MacKenzie et al. 2009), and 36%–42% aa identity with the MucBP domain (fragments 187–294) of the protein LBA1460 from L. acidophilus NCFM. Numerous MUB homologs and proteins containing the MucBP domain have been identified in Lactobacilli naturally located in the gut niche, and they have been shown to play important roles in the adhesion of probiotic strains to intestinal mucins (Ossowski et al. 2011; Etzold et al. 2014; Jensen et al. 2014) and epithelial cells (Call and Klaenhammer, 2013; Jensen et al. 2014). In this study, we found that N506_1778 and its homologs are also present in Lactobacilli naturally located in the vaginal niche. We then compared the homologs of protein N506_1778 in all 10 known Lactobacillus gasseri genomes (7 strains from the human vagina) (Table 3). Interestingly, all known Lactobacillus gasseri genomes contained homologs of N506_1778, with aa identity ranging from 79-98% (Table 3). The high similarity to other homologous proteins in *Lactobacillus gasseri* suggests that N506_1778 may play an important role in the species' adaptation to different host niches. Therefore, double crossover was used to knock out the N506_1778 gene, and the adhesion ability of the mutant to different epithelial cells was evaluated.

[0168] Table 3

[0169] Homology of N506_1778 with other genes in Lactobacillus gasseri

[0170]

[0171]

[0172] The N506_1709 gene in *Lactobacillus gasseri* DSM 14869 consists of a 4.371 kb sequence encoding a large surface protein of 1456 amino acids with a predicted molecular weight of 158.9 kDa. This protein contains a YSIRK signal peptide, an N-terminal region (amino acids 42 to 1233), an internal repeat region (amino acids 892 to 1372), and a C-terminal LPQTG anchoring motif. The internal repeat region contains three repeats (the first being a partial repeat), which showed similarity to the rib / α-like repeat domain (PF08428) in Pfam analysis. Figure 7 C). Following BLASTP analysis, the inventors found that N506_1709 shared high sequence identity (99%) with the hypothetical protein (LJCM1025_14810) from *Lactobacillus gasseri* LJCM1025, but less than 10% identity with other proteins in the database. The last 600 amino acids, containing rib / α-like repeat regions, showed 34%-48% amino acid identity with surface proteins from *Lactobacillus johnsonii* and *Lactobacillus gasseri*. Figure 7 C). Rib / α-like repeat domains have also been found in various cell surface proteins of *Lactobacillus*, suggesting that proteins containing this domain may promote bacterial adhesion to stratified squamous epithelial cells (Edelman et al. 2012; Stallhammar-Carlemalm et al. 1999). Because the protein sequence characteristics of N506_1709 suggest it may be a novel putative adhesion protein that promotes the binding of *Lactobacillus gasseri* DSM14869 to vaginal epithelial cells, the encoding gene was deleted via double cross-recombination.

[0173] Construction of N506_1778 and N506_1709 knockout mutants. For example... Figure 11 As shown in A and B, the N506_1778 and N506_1709 mutants did not express the mRNA of genes N506_1778 and N506_1709, while the mRNA expression levels of the corresponding genes in the complementary strains were restored to wild-type. These results indicate that genes N506_1778 and N506_1709 were successfully deleted from the genome of *Lactobacillus gasseri* DSM14869.

[0174] N506_1778-mediated binding of Lactobacillus gasseri DSM 14869 to Caco-2, HeLa, and human vaginal cells.

[0175] The growth rate of the N506_1778 mutant strain was not altered under the growth conditions used in this study. Figure 14The effects of the N506_1778 mutation were confirmed by evaluating its ability to adhere to Caco-2, HeLa, and vaginal epithelial cells in vitro. Figure 12A As shown in -C, compared with the wild-type strain, the N506_1778 mutation resulted in significantly reduced adhesion to Caco-2 (42%, p<0.01), HeLa (32%, p<0.001), and human vaginal cells (32%, p<0.01). Complementation with pNZe-N506_1778 restored wild-type level adhesion to vaginal cells. Figure 12C These results indicate that N506_1778 is an important cell surface protein involved in the adhesion of Lactobacillus gasseri DSM 14869 to epithelial cells of different hosts.

[0176] N506_1709-mediated binding of *Lactobacillus gasseri* DSM 14869 to human vaginal cells, but not to Caco-2 and HeLa cells. To determine the contribution of N506_1709 to bacterial adhesion, an N506_1709 knockout mutant was constructed. The mutant strain showed the same growth rate as the wild type. Figure 14 First, the adhesion of this mutant to the columnar epithelial cell lines Caco-2 and HeLa was assessed. Figure 6 As shown in Figures A and B, the N506_1709 mutant strain *Lactobacillus gasseri* DSM14869-ΔN506_1709 did not show a significant difference in adhesion to Caco-2 and HeLa cells compared to the wild-type strain. Since N506_1709 includes a rib / α-like repeat domain, it has been shown to be involved in binding to layered squamous epithelial cells (Edelman et al. 2012), and the inventors subsequently investigated whether N506_1709 plays a role in adhesion to human vaginal cells (a type of layered squamous epithelial cell). Figure 12C As shown, compared with the wild type, the N506_1709 mutant exhibited a significant decrease of approximately 42% in adhesion ability to human vaginal cells (p<0.001). To confirm the genotype-phenotype relationship of the N506_1709 gene, the mutant strain was subsequently supplemented by reintroducing the N506_1709 gene. The complement strain *Lactobacillus gasseri* DSM 14869-ΔN506_1709 / pNZ8048-N506_1709 showed partial recovery of adhesion levels. Figure 12C This indicates that N506_1709 mediates tissue-specific adhesion to layered squamous epithelial cells of the vagina.

[0177] Overexpression of N506_1709 in *Lactococcus lactis* NZ9000 increases adhesion to vaginal epithelial cells. To confirm tissue-specific adhesion of N506_1709, the protein N506_1709 was overexpressed in *Lactococcus lactis* NZ9000 using the pNZ8048 vector and its nisin-induced expression system. *Lactococcus lactis* NZ9000 transformed with the empty plasmid pNZ8048 was used as a control. After nisin-induced expression of N506_1709 in *Lactococcus lactis* NZ9000, the overexpressing strain showed a 5000-fold increase in mRNA transcription compared to the control. Figure 13 A). Furthermore, the overexpressing strain showed a 2.8-fold increase in adhesion to vaginal cells (p<0.001). Figure 13 B) confirmed that N506_1709 plays a role in the adhesion ability of Lactobacillus gasseri DSM 14869 to human vaginal epithelium.

[0178] discuss

[0179] Lactobacillus gasseri is one of the major species isolated from the vaginal microbiota (Pendharkar et al. 2013; Ravel et al. 2011), and various health benefits have been reported in strains of Lactobacillus gasseri (Marcotte et al. 2017; Parolin et al. 2015). However, the molecular mechanisms promoting these health effects, such as adhesion factors that enable optimal adhesion of Lactobacillus to this niche, are generally not well understood. In this study, we genetically identified and functionally analyzed three genes that may contribute to the adhesion of the probiotic strain Lactobacillus gasseri DSM 14869 to vaginal epithelial cells.

[0180] First, the function of the EPS gene cluster identified in *Lactobacillus gasseri* DSM 14869 was evaluated by mutations in the N506_0400 gene encoding the putative initiating glycosyltransferase. According to the literature, the gene encoding the initiating glycosyltransferase is highly conserved (Jolly and Stingele, 2001), and the initiating glycosyltransferase plays a crucial role in the first step of EPS biosynthesis by transferring the first sugar to the UndP-lipocarrier (Lebeer et al. 2009). Mutations in the genes encoding the initiating glycosyltransferase in *Lactobacillus johnsonii*, *Lactobacillus rhamnosus*, or *Lactobacillus paracasei* eliminated or reduced the production of heteropolysaccharides (Lebeer et al. 2009; Horn et al. 2012). (et al. 2016). In this study, the increased auto-aggregation ability and decreased expression of surface-associated polysaccharides in the N506_0400 mutant strain compared to the wild type also indicated the crucial role of galactosyltransferase N506_0400 in EPS biosynthesis in *Lactobacillus gasseri* DSM 14869. Furthermore, when grown in MRS medium, the EPS mutant of *Lactobacillus gasseri* DSM 14869 showed a significant increase in biofilm formation, while the complement strain partially restored biofilm formation ability to that of the wild type (…). Figure 9C Biofilm formation is considered an important surface characteristic of probiotics, involved in their beneficial effects on the host (Younes et al. 2012; Jones & Versalovic, 2009). This suggests that the ability to form a biofilm may be related to sorting enzyme A-dependent proteins (SDPs). For example, the EPS mutant of *Lactobacillus rhamnosus* GG showed a significant increase in biofilm formation, presumably due to increased exposure to cell surface adhesins after EPS removal (Lebeer et al. 2009; Lebeer et al. 2012). Malik et al. (2013) also reported that biofilm formation in *Lactobacillus plantarum* CMPG 5300 strain may be due to SDPs, as the srtA mutant of this strain lost its biofilm-forming ability. Therefore, in this study, the increased biofilm-forming ability of the EPS mutant strain may also be due to exposure to SDPs.

[0181] Furthermore, the role of EPS in adhesion was investigated using Caco-2, HeLa, and vaginal epithelial cells. EPS mutants significantly increased adhesion to Caco-2 colon cancer cells and HeLa cervical cancer cells, but slightly decreased adhesion to vaginal epithelial cells. The increased adhesion to Caco-2 and HeLa cells may be attributed to better adhesin exposure due to the absence of EPS. These data are consistent with studies on *Lactobacillus rhamnosus* GG, which showed that deprivation of galactose-rich EPS increased adhesion to Caco-2 cells, likely due to increased adhesin exposure (Lebeer et al. 2009), and further studies on the specific adhesive properties of *Lactobacillus rhamnosus* GG fimbriae confirmed this “shielding hypothesis” (Lebeer et al. 2012). Similar results have also been reported in *Lactobacillus johnsonii* NCC533 (Denou et al. 2008), *Lactobacillus rhamnosus* E / N (Polak-Berecka et al. 2014), and *Lactobacillus plantarum* Lp90 strain (Lee et al. 2016). However, the EPS mutant showed a slight decrease (30%) in its adhesion to vaginal epithelial cells, suggesting that EPS may promote the adhesion of *Lactobacillus gasseri* DSM 14869 to vaginal epithelial cells. The effect of EPS on adhesion is strain-specific and may be related to differences in EPS structural characteristics and the cell surface properties of the strain. For example, mutations in the cps family in *Lactobacillus plantarum* strains WCFS1 and SF2A35B had no significant effect on the adhesion of Caco-2 cells (Lee et al. 2016). et al. (2016) reported that the presence of EPS-SJ P2 increased adhesion to Caco-2 cells, which may be attributed to the molecular structure of the EPS-SJ P2 matrix. Therefore, the increased specificity of Lactobacillus gasseri DSM14869 via EPS for vaginal cell adhesion may be due to differences in the molecular structure of EPS compared to Caco-2 and HeLa cells, or differences in receptors within vaginal cells. How EPS promotes bacterial adhesion to vaginal cells requires further investigation. To our knowledge, this is the first report demonstrating that EPS from Lactobacillus vaginalis can provide tissue-oriented adhesion. This will contribute to a better understanding of its specific contribution to probiotic-host interactions and its role in adapting to this vaginal niche.

[0182] Increased adhesion of some lactobacilli to the intestinal mucosa has been thought to be mediated by cell surface proteins with mucus-binding capabilities (Kankainen et al. 2009; Rojas et al. 2002; Ossowski et al. 2011; Jensenet et al. 2014). The N506_1778 protein, containing two repeats homologous to the MucBP domain, is the only predicted cell wall anchoring protein, including the MucBP-like domain found in *Lactobacillus gasseri* DSM14869. Our results indicate that the N506_1778 protein can promote the adhesion of *Lactobacillus gasseri* DSM 14869 to Caco-2, HeLa, and human vaginal epithelial cells. Homologs of N506_1778 were found in known *Lactobacillus gasseri* strains isolated from different ecological niches (Table 3), suggesting that N506_1778 is an important cell surface protein for *Lactobacillus gasseri* species to adapt to different host ecological niches. This is the first report that proteins with MucBP-like domains are also involved in the adhesion of vaginal epithelial cells.

[0183] N506_1709 is another important cell surface protein mediating the adhesion of *Lactobacillus gasseri* DSM 14869 to vaginal mucosal cells. It is a newly described sorting enzyme-dependent adhesin exhibiting specific binding to stratified squamous epithelial cells. Interestingly, N506_1709 differs from previously characterized lactobacillus adhesins such as Lsp, Mub, and mucus-binding factor (MBF) (Walter et al. 2005; Buck et al. 2005; von Ossowski et al. 2011) because it lacks the MuBP domain and instead contains three repeating regions homologous to Rib / α-like repeat sequences. Rib and α proteins were first identified in streptococci and are thought to be involved in pathogen adhesion and biofilm formation (Michel et al. 1992; et al. 1996; –Carlemalm et al. 1999). Subsequently, proteins homologous to Rib / α-like repeats were also reported in vaginal lactobacilli, such as protein Rlp in *Lactobacillus fermentum* and LEA in *Lactobacillus curvatureii* (Turner et al. 2003; Edelman et al. 2012). These proteins with Rib / α-like repeat domains in lactobacilli are thought to mediate binding to the host's layered squamous epithelial lining (Edelman et al. 2012; Turner et al. 2003). In this study, the N506_1709 mutant showed significantly reduced adhesion to vaginal epithelial cells (layered squamous epithelial cells), but no reduction in adhesion to colon cancer cells and cervical cancer cells (columnar epithelial cells). This suggests that the N506_1709 protein provides tissue tropism for *Lactobacillus gasseri* DSM 14869, possibly determined by the presence of different receptors on the cell membrane of vaginal epithelial cells. Overexpression of N506_1709 in *Lactococcus lactis* significantly improved the adhesion of *Lactococcus lactis* to vaginal epithelial cells, further confirming the adhesive ability of N506_1709 to vaginal epithelial cells. In summary, N506_1709 is an important surface protein mediating the adhesion of *Lactobacillus gasseri* to human vaginal epithelium, which can promote bacterial colonization in the host and may have ecological importance.

[0184] In summary, this report identified and functionally analyzed three cell surface molecules, including EPS, N506_1778, and N506_1709, as important adhesion factors for *Lactobacillus gasseri* DSM 14869 in vaginal adhesion. To our knowledge, this is the first report demonstrating the role of EPS in vaginal *Lactobacillus* strain adhesion, and proteins with MucBP-like domains may also be involved in vaginal epithelial adhesion. Furthermore, N506_1709, labeled as *Lactobacillus vaginal epithelial adhesin* (LVEA), may be a novel adhesin that specifically mediates cell-to-layer squamous epithelial cell binding. These results provide important new information on the molecular mechanisms of *Lactobacillus* adhesion and tissue orientation to various host mucosal surfaces, and will help us screen for better probiotic candidates in the future. Further research is needed to determine the specific receptors for EPS and LVEA on vaginal epithelial cells and the functional domains of LVEA.

[0185] Materials and methods

[0186] Bacterial strains, plasmids, and growth conditions. Table 4 lists the bacterial strains and plasmids used in this study.

[0187] Table 4. Bacterial strains and plasmids used in this study

[0188]

[0189]

[0190] a Km r Kanamycin resistance; Cm r Chloramphenicol resistance; Em r Erythromycin resistance.

[0191] Use shaken at 37°C to replenish 25 μg / ml. -1 Escherichia coli VE7108 (Mora et al. 2004) strain was cultured in Luria broth containing kanamycin. Lactic acid bacteria strains were statically grown in MRS broth under anaerobic conditions at 37°C. When necessary, the following antibiotic was added: Escherichia coli, 10 μg / ml. -1 Chloramphenicol (Cm) and 300 μg ml -1 Erythromycin (Em); Lactobacillus transformant, 10 μg / ml -1 Cm and 5μg ml -1 Em.

[0192] DNA manipulation. Standard DNA protocols were used for DNA manipulation in *E. coli* (Sambrook et al. 1989). Plasmid DNA was extracted from *E. coli* using the QIAGEN Miniprep Spin Kit (Qiagen, Hilden, Germany). *Lactobacillus gasseri* DNA was isolated using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany). Primers (Table 5) were synthesized by Eurofins Genomics (Ebersberg, Germany). Phusion high-fidelity DNA polymerase (Finnzymes / Thermo Fisher Scientific, Espoo, Finland) was used for amplification of *Lactobacillus gasseri* genomic DNA, and GoTaq DNA polymerase (Promega, Fitchburg, WI, USA) was used for colony PCR. PCR products were purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). Restriction endonucleases were supplied by Thermo Scientific, and T4 DNA ligase was from Invitrogen (Carlsbad, CA). All procedures were performed according to the manufacturer's instructions. As previously described, the plasmid was transformed into Lactobacillus gasseri DSM 14869 via electroporation (DeKeersmaecker et al. 2006).

[0193] Table 5. List of primers used in this study

[0194]

[0195]

[0196]

[0197] a Restriction sites are underlined in the primer sequence.

[0198] EPS (N506_0400), N506_1778, and N506_1709 knockout mutants were constructed via bimodal recombination. In this study, the reproducible plasmid pINTZrec (unpublished data) was used... Figure 15A This plasmid is used to mediate homologous recombination. It comprises two six sites and a β-recombinase, which specifically catalyzes recombination between the two six sites flanking the antibiotic resistance gene and the DNA to be integrated. Expression of the β-recombinase is controlled by the sakacin-inducible promoter. To delete the N506_0400 gene from the genome of *Lactobacillus gasseri* DSM 14869, approximately 1.0 kb upstream and downstream fragments flanking the 5' and 3' ends of the N506_0400 gene were amplified by PCR using primers 400 upstream-F / R and 400 downstream-F / R, respectively (Table 5). The resulting amplicons were ligated using an overlap extension strategy with primer pair 400 upstream-F / downstream-R (Table 5). The PCR products obtained by digestion with Sac I and Nhe I were ligated into a similarly digested pINTZrec plasmid and transformed into electrocompetent *E. coli* VE7108 to obtain the final plasmid construct pINTZrec-N506_0400. pINTZrec-N506_0400 was then electroporated into competent *Lactobacillus gasseri* DSM 14869 cells prepared according to De Keersmaecker et al. (2006). For recombinant-activated gene expression, β-recombinase gene expression was induced using sakacin. In short, the transformed *Lactobacillus gasseri* strains were inoculated into MRS medium and grown to OD0.05. 600 At approximately 0.5, add 100 ng / ml -1 Sakacin was used for overnight induction. Serial dilutions of the induced culture were inoculated into MRS agar plates (containing 10 μg / ml). -1 Cm and 100ng ml - 1The bacteria were anaerobic and grown for 48 hours. To obtain reproducible plasmids that had been excised and single crossover integration, single colonies with the Cm gene but without the repA gene were selected and further confirmed by PCR using seq1-F / INTZ-R and INTZ-F / seq1-R primers (Table 5). Single crossover strains were grown in 3 ml of antibiotic-free MRS medium and subcultured twice daily for 2 days. Subsequently, the bacterial cultures were diluted and inoculated onto antibiotic-free MRS plates for 48 hours to obtain single colony isolates, which were then replicated and inoculated onto plates with 10 μg / ml of sakacin. -1 On Cm MRS plates. Non-antibiotic resistant colonies were examined using specific primers seq1-F / seq1-R to obtain the N506_0400 gene deletion strain, Lactobacillus gasseri DSM14869-ΔN506_0400.

[0199] To remove the N506_1778 and N506_1709 genes from the genome of *Lactobacillus gasseri* DSM 14869, the same EPS knockout method was used. Briefly, approximately 1.0 kb upstream and downstream fragments flanking the 5' and 3' ends of the N506_1778 and N506_1709 genes were amplified by PCR using primers 1778 upstream-F / R and 1778 downstream-F / R, and 1709 upstream-F / R and 1709 downstream-F / R, respectively. The resulting amplicons were ligated using an overlap extension strategy with primer pairs 1778 upstream-F / downstream-R and 1709 upstream-F / downstream-R (Table 5). PCR products obtained from Sac I and Bam HI digestion were ligated into a similarly digested pINTZrec plasmid and transformed into *E. coli* VE7108 to obtain plasmid constructions pINTZrec-N506_1778 and pINTZrec-N506_1709. Plasmids pINTZrec-N506_1778 and pINTZrec-N506_1709 were electroporated into DSM14869, and the transformed strains were induced by sakacin. Single cross-integrated colonies were screened by PCR using seq2-F / INTZ-R and INTZ-F / seq2-R primers for N506_1778, and seq3-F / INTZ-R and INTZ-F / seq3-R primers for N506_1709. Finally, the double cross-integrated strain was examined using specific primers seq2-F / seq2-R to obtain a strain with the N506_1778 gene deletion, named *Lactobacillus gasseri* DSM14869-ΔN506_1778, and the double cross-integrated strain was examined using specific primers seq3-F / seq3-R to obtain a strain with the N506_1709 gene deletion, named *Lactobacillus gasseri* DSM14869-ΔN506_1709.

[0200] Complementation: Plasmid Construction and Transformation. The plasmid pNZe-Rec was used as the starting material to achieve complementation of the N506_0400 gene in strain *Lactobacillus gasseri* DSM14869-ΔN506_0400. The N506_0400 gene does not have its own promoter; it shares the promoter of the EPS operon. Therefore, the promoter of the EPS gene cluster was amplified using primers EPS-promoter-F / R (Table 5), and the N506_0400 gene was amplified using primers N506_0400-F / R. The promoter and N506_0400 gene were ligated by overlap extension using primer pair EPS-promoter-F / N506_0400-R, then digested with Kpn I and Hin dIII and ligated into a similarly digested pNZe-Rec to produce pNZe-N506_0400. The plasmid pNZe-N506_0400 was electroporated into Lactobacillus gasseri DSM14869-ΔN506_0400 to generate an Em-sensitive strain, Lactobacillus gasseri DSM14869-ΔN506_0400 / pNZe-N506_0400.

[0201] The plasmid pNZe-N506_0400 was used to complement the gene N506_1778 in the Lactobacillus gasseri DSM14869-ΔN506_1778. The gene N506_1778 and its promoter were amplified by PCR using specific primers N506_1778-F / R. The PCR product was digested with Kpn I and Bam HI and ligated into pNZe-N506_0400 digested with Kpn I and Bgl II (Bam HI and Bgl II are isoskeletal enzymes) to produce pNZe-N506_1778. Plasmid pNZe-N506_1778 was electroporated into Lactobacillus gasseri DSM 14869-ΔN506_1778 to obtain an Em-sensitive strain, Lactobacillus gasseri DSM 14869-ΔN506_1778 / pNZe-N506_1778.

[0202] Plasmid pNZ8048 was used to complement the gene N506_1709 in the complementary strain *Lactobacillus gasseri* DSM 14869-ΔN506_1709. The N506_1709 gene was ligated to pNZ8048 in two steps. First, the gene N506_1709 (approximately 2 kb) and the first part of its promoter were amplified using primers N506_1709-F1 / R1. The PCR product was digested with Bgl II and Kpn I and ligated to similarly digested pNZ8048, producing plasmid pNZ8048-N506_1709-1. Then, the second part (approximately 2.3 kb) of the N506_1709 gene was amplified using primers N506_1709-F2 / R2 and digested with Kpn I and Sac I, ligated into a similarly digested pNZ8048-N506_1709-1 to generate pNZ8048-N506_1709. The plasmid pNZ8048-N506_1709 was electroporated into *Lactobacillus gasseri* DSM14869-ΔN506_1709 to obtain the Cm-sensitive strain, *Lactobacillus gasseri* DSM14869-ΔN506_1709 / pNZ8048-N506_1709.

[0203] Construction of an overexpression construct for N506_1709 in *Lactococcus lactis* NZ9000. To heterologously express N506_1709 in *Lactococcus lactis*, the nisin-inducible vector pNZ8048 was used. The N506_1709 gene from *Lactobacillus gasseri* DSM14869 was amplified using primers N506_1709Re-F / R (Table 5) and subsequently cloned into the pNZ8048 vector, generating the plasmid pNZ8048-N506_1709Re. The plasmid pNZ8048-N506_1709Re was used to transform competent *Lactococcus lactis* NZ9000 cells, producing the strain *Lactococcus lactis* NZ9000 / pNZ8048-N506_1709Re.

[0204] RNA extraction and quantitative real-time PCR (qRT-PCR) were performed. RNA was extracted from 10 cells grown in the logarithmic growth phase using the RNeasy Mini kit (Qiagen). 9 Total RNA was extracted from bacteria. Reverse transcription was performed using the QuantiTect Reverse Transcription Kit (Qiagen) containing 1 μg of total RNA as a template. qRT-PCR was performed using the SYBR Green Assay Kit (Qiagen). Specific primers were designed using PrimerPremier 5 software (Table 5), with the internal gene 16S rRNA used as a reference. ΔΔC was used... T The method (Schmittgen & Livak, 2008) was used to calculate relative gene expression.

[0205] Automated aggregation analysis. Automated aggregation analysis was performed using some modifications as previously reported (LecceseTerraf1 et al. 2014). In short, bacteria were grown overnight (approximately 16 h) in MRS. The cultures were centrifuged and washed twice with phosphate-buffered saline (PBS) (pH 7.2), then resuspended in PBS to an OD of 1.5. 600 After incubation at room temperature for 5 hours, the OD of the supernatant was measured. 600 The percentage of automatic aggregation is calculated using the following expression: Automatic aggregation (%) = (1 - (OD) / ... 600 5h / OD 600 0h))×100, where OD 600 5h represents the absorbance at time point 5 hours, and OD 600 0h indicates the absorbance at 0h.

[0206] Biofilm formation assay. Biofilm formation was performed using some modifications as previously described (Lebeer et al. 2007). Briefly, biofilms were grown in MRS medium in 96-well polystyrene microplates at 37°C for 72 h. The wells were then washed three times with PBS and stained with 0.1% crystal violet for 30 min. Excess staining was rinsed with water and the wells were air-dried (1 h). The stain bound to the adherent cells was extracted with 200 μl of 30% glacial acetic acid. The OD of 135 μl from each well was measured. 570 The experiment was repeated three times, with eight replicates each time. Additionally, sterile MRS medium was used as a negative control.

[0207] Rinse each well with 200ml of distilled water to remove excess dye.

[0208] Transmission electron microscopy (TEM). Cells of *Lactobacillus gasseri* DSM 14869, EPS mutants, and complement strains were grown overnight in MRS, and the EPS layer present on the surface of *Lactobacillus gasseri* strains was analyzed by TEM as previously described (Alvarez et al. 2015). Relative thickness is expressed as a percentage relative to WT (set as 100%). Thickness was assessed for 10 cells in each group, and the mean ± SD of the thickness of each cell was determined.

[0209] Assay for Caco-2 and HeLa cell adhesion. The cells were supplemented with 10% fetal bovine serum (FBS) and 100 IU / ml. -1 Penicillin G and 100 μg ml -1 Caco-2 and HeLa cells were routinely grown in streptomycin-treated DMEM medium. Adhesion assays were performed using some modifications as previously described (Lee et al. 2016). In short, at 10... 5Cells were seeded in 24-well plates at a concentration of [number] cells / well and cultured for 72 h until confluence. *Lactobacillus gasseri* DSM 14869 was allowed to grow for 18 h and washed twice with PBS. 7 CFU ml -1 The culture medium was resuspended in antibiotic-free Dulbecco modified Eagle medium (DMEM). 0.8 ml of bacterial culture was added to each well and incubated for 2 h. The wells were washed three times with PBS to remove unadhered bacteria. After washing, 0.2 ml of trypsin-EDTA (Invitrogen) was added to the wells to detach the cells, followed by 0.6 ml of antibiotic-free DMEM to stop trypsin digestion. Serial dilutions were prepared and inoculated onto MRS agar plates to count the number of adherent bacteria. The adhesion rate was calculated as the percentage of bacteria adhering to Caco-2 or HeLa cells relative to the total number of bacteria added to the wells. All adhesion experiments were performed in triplicate and repeated three times.

[0210] Assay for vaginal epithelial cell (VEC) adhesion. The protocol was approved by the Stockholm Ethics Committee (Regionala). Approved by i Stockholm (License No.: 2018 / 1090 / 31). Informed consent was obtained from participants before the start of the study. VEC cells were collected from four healthy volunteer donors by gently scraping the vaginal mucosa surface with a sterile swab and suspending them in 10 ml of DMEM medium. Cells were washed three times with 10 ml of DMEM and centrifuged at 800 × g for 5 min to remove protozoa. Cells were adjusted to 10⁻¹⁰ cells in DMEM using a hemocytometer. 5 cells / ml -1 Lactobacillus gasseri DSM 14869 harvested from 18h culture was washed twice with PBS (pH 7.4) and resuspended in DMEM medium to obtain 5 × 10⁻⁶ samples. 7 CFU ml -1 The final concentration. In the overexpression adhesion assay, Lactococcus lactis strains were inoculated into OD. 600 Approximately 0.5 and using 10 ng / mL -1 Nisin (50 IU mL) -1 Induce with Sigma for 1.5 h. Then harvest the induced culture as described above.

[0211] An equal volume (400 μl) of vaginal epithelial cells and *Lactobacillus* or *Lactococcus lactis* were mixed and incubated at 37°C for 2 h. After incubation, the cells were washed five times with PBS to remove non-adhesive bacteria. After the final centrifugation, the cell clumps were transferred to a microscope slide, dried, fixed with methanol, and stained with 0.1% crystal violet. Each sample was performed in triplicate, and 100 randomly selected cells from each replicate were examined under an optical microscope in oil immersion. Results are expressed as the bacterial count per cell. VECs not incubated with *Lactobacillus* or *Lactococcus lactis* were included as a negative control.

[0212] Statistical analysis. Data are expressed as mean ± SD. Significant differences between means were determined using SPSS 20 by one-way ANOVA followed by the Duncan test (P < 0.05). All experiments were performed in triplicate and repeated three times.

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accaagctag ctgtatcact tttggtattg a 31 <210> twenty three <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Seq1-F <400> twenty three attgggttct aacagaatgc gt 22 <210> twenty four <211> twenty three <212> DNA <213> Artificial sequence <220> <223> INTZ-R <400> twenty four ttctccccct aataattctg cag 23 <210> 25 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> INTZ-F <400> 25 ggtttttata ttacagctcc aag 23 <210> 26 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Seq1-R <400> 26 aatcctgaat cactttcggt tt 22 <210> 27 <211> 30 <212> DNA <213> Artificial sequence <220> <223> 1778 upstream-F <400> 27 caacggagct ccgaaactaa tggcatcaat 30 <210> 28 <211> 43 <212> DNA <213> Artificial sequence <220> <223> 1778 upstream-R <400> 28 gtttttggtg cagagaaatt tatatataaa ataacgattt tgt 43 <210> 29 <211> 47 <212> DNA <213> Artificial sequence <220> <223> 1778 downstream-F <400> 29 ttgtatagga gaaacaaggt ttcttgataa taaataacat taatagc 47 <210> 30 <211> 35 <212> DNA <213> Artificial sequence <220> <223> 1778 downstream-R <400> 30 ctttaaggat ccaaaagaag aagctagaaa ggctt 35 <210> 31 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Seq2-F <400> 31 cacataatac cagcagtcaa cgaa 24 <210> 32 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Seq2-R <400> 32 gtgctccaag tagtatcata gcgat 25 <210> 33 <211> 34 <212> DNA <213> Artificial sequence <220> <223> 1709 upstream-F <400> 33 ttcctggagc tcaaacttta ttttgttctg ccaa 34 <210> 34 <211> 43 <212> DNA <213> Artificial sequence <220> <223> 1709 upstream-R <400> 34 atgttattta ttatcaagaa accttgtttc tcctatacaa tgt 43 <210> 35 <211> 47 <212> DNA <213> Artificial sequence <220> <223> 1709 downstream-F <400> 35 ttgtatagga gaaacaaggt ttcttgataa taaataacat taatagc 47 <210> 36 <211> 35 <212> DNA <213> Artificial sequence <220> <223> 1709 downstream-R <400> 36 ctttaaggat ccaaaagaag aagctagaaa ggctt 35 <210> 37 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Seq3-F <400> 37 ggctaaaaac agactccatc aatc 24 <210> 38 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Seq3-R <400> 38 agcccgttct ttcttataac tttaa 25 <210> 39 <211> 36 <212> DNA <213> Artificial sequence <220> <223> EPS - Promoter - F <400> 39 aattaggtac cacactgtaa aaataaataa gatcct 36 <210> 40 <211> 40 <212> DNA <213> Artificial sequence <220> <223> EPS-starter-R <400> 40 ttaacctctt gtgccatcat tttattcctc ttttattttt 40 <210> 41 <211> 40 <212> DNA <213> Artificial sequence <220> <223> N506_0400-F <400> 41 aaaaataaaa gaggaataaa atgatggcac aagaggttaa 40 <210> 42 <211> 33 <212> DNA <213> Artificial sequence <220> <223> N506_0400-R <400> 42 cttttaagct taatacgcac tatttggatg aat 33 <210> 43 <211> 36 <212> DNA <213> Artificial sequence <220> <223> N506_1778-F <400> 43 tacatcggta ccaaaatatg cggtatgtat ttatcg 36 <210> 44 <211> 38 <212> DNA <213> Artificial sequence <220> <223> N506_1778-R <400> 44 ggtgcaggat ccttaattct ttttctttcg tttaagtt 38 <210> 45 <211> 38 <212> DNA <213> Artificial sequence <220> <223> N506_1709-F1 <400> 45 aagacagatc tcgtaattaa attgatcaag tacattat 38 <210> 46 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> N506_1709-R1 <400> 46 acaaaggtac caccagattc cat 23 <210> 47 <211> 25 <212> DNA <213> Artificial sequence <220> <223> N506_1709-F2 <400> 47 ctggtggtac ctttgtttca aaagt 25 <210> 48 <211> 39 <212> DNA <213> Artificial sequence <220> <223> N506_1709-R2 <400> 48 attatgagct cttatctaat tcggtgtttt cttctactt 39 <210> 49 <211> 37 <212> DNA <213> Artificial sequence <220> <223> N506_1709Re-F <400> 49 gagaaaccat ggatgctatc taaaaataat tttcatg 37 <210> 50 <211> 51 <212> DNA <213> Artificial sequence <220> <223> N506_1709 Re-R <400> 50 ttaatgagct cttacgcttc cggttctcta attcggtgtt ttcttctact t 51 <210> 51 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Lactobacillus gasseri 16S-F <400> 51 acccttgtca ttagttgcca tca 23 <210> 52 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Lactobacillus gasseri 16S-R <400> 52 gcttctcgtt gtaccgtcca tt 22 <210> 53 <211> 26 <212> DNA <213> Artificial sequence <220> <223> 1778-F <400> 53 agaccttaga gagcaagtca ttatcg 26 <210> 54 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> 1778-R <400> 54 ttggttatta ggaagttcgt cgtt 24 <210> 55 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> 1709-F <400> 55 attggaacga tttgaagagc g 21 <210> 56 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> 1709-R <400> 56 gaatcagtag tgtgggaacc gac 23 <210> 57 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Lactococcus lactis 16S-F <400> 57 tcgtgtcgtg agatgttggg t 21 <210> 58 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Lactococcus lactis 16S-R <400> 58 gtcataaggg gcatgatgat ttg 23

Claims

1. A circular DNA vector comprising: (a) An optional marker gene sequence, wherein the marker gene sequence is operatively linked to a first promoter sequence. (b) A multiple cloning site, wherein the multiple cloning site comprises a gene-targeting sequence, wherein the target sequence comprises flanking sequences comprising at least 400 consecutive nucleotides having at least 95% sequence identity with a corresponding region of the target region of the host cell genome. (c) A sequence encoding a site-specific recombinase, wherein the sequence is operatively ligated to a second promoter sequence, wherein the second promoter is inducible. (d) Copy the subsequence, (e) Two target sites for the site-specific recombinase. The vector comprises a first region, the two target sites for the site-specific recombinase forming flanks on opposite sides of the first region, and the region comprises (a) and (b), provided that (c) and (d) are not within the first region; and The two target sites for the site-specific recombinase are oriented such that the product of site-specific recombination between the two target sites for the site-specific recombinase is a first circular DNA product comprising (a) and (b) and a second circular DNA product comprising (c) and (d).

2. The circular DNA vector of claim 1, wherein the optional marker is an antibiotic resistance gene.

3. The circular DNA vector according to claim 1, wherein the site-specific recombinase is a site-specific serine recombinase.

4. The circular DNA vector according to claim 1, wherein the site-specific recombinase is selected from the group consisting of β-recombinase, Cre-recombinase, FLP-recombinase and PhiC31 integrase.

5. The circular DNA vector according to claim 1, wherein the replicon sequence is a prokaryotic replicon sequence.

6. The circular DNA vector according to claim 1, wherein the replicon sequence encodes the origin of replication (ori) and the replication initiation protein (Rep protein).

7. The circular DNA vector according to claim 1, wherein the replicon sequence is a replicon sequence that allows the vector to replicate in a prokaryotic host cell.

8. The circular DNA vector of claim 1, wherein the replicon sequence is a replicon sequence that allows the vector to replicate in Lactobacilli or Bifidobacteria.

9. The circular DNA vector of claim 1, wherein the replicon sequence is a replicon sequence that allows the vector to be replicated in Escherichia coli (E. coli).

10. The circular DNA vector of claim 1, wherein the replicon sequence is a replicon sequence that allows the vector to replicate in Escherichia coli and another prokaryotic host cell selected from the group consisting of Lactobacillus and Bifidobacterium.

11. The circular DNA vector of claim 1, wherein the replicon sequence is a replicon sequence that allows the vector to replicate in at least one host cell selected from the group consisting of: *Lactobacillus gasseri*, *Lactobacillus rhamnosus*, *Lactobacillus paracasei*, *Lactobacillus plantarum*, *Lactobacillus acidophilus*, *Lactobacillus fermentum*, *Lactobacillus crispatus*, *Lactobacillus jensenii*, *Lactobacillus vaginalis*, *Lactobacillus iners*, *Lactobacillus reuteri*, *Lactobacillus casei*, *Lactobacillus bulgaricus*, and *Lactobacillus curvilinearus*. Lactobacillus curvatus, Lactobacillus delbrueckii, and Lactobacillus johnsonii.

12. The circular DNA vector according to claim 1, wherein the second promoter sequence is an inducible prokaryotic promoter.

13. The circular DNA vector according to claim 1, wherein the second promoter sequence is an IPTG-inducible promoter.

14. The circular DNA vector according to claim 1, wherein the second promoter sequence is a synthetically inducible promoter.

15. The circular DNA vector according to claim 14, wherein the synthetic inducible promoter is selected from the group consisting of tetracycline inducible promoter, D-xylose inducible promoter and lactose inducible promoter.

16. The circular DNA vector according to claim 1, wherein the second promoter sequence is a bacteriocin-inducible promoter.

17. The circular DNA vector according to claim 16, wherein the bacteriocin-inducible promoter is a sakacin-inducible promoter or a nisin-inducible promoter.

18. A method for introducing recombination between a circular DNA vector and a target region of a lactobacillus host cell genome, the method comprising the steps of: (i) Introducing a circular DNA vector containing a gene-targeting sequence according to claim 1 into a Lactobacillus host cell, wherein the target sequence includes flanking sequences comprising at least 400 consecutive nucleotides having at least 95% sequence identity with a corresponding region of the target region of the host cell genome. (ii) Inducing the expression of a site-specific recombinase encoded by the circular DNA vector, and allowing site-specific recombination between target sites of the site-specific recombinase to produce a first circular DNA product comprising (a) and (b) and a second circular DNA product comprising (c) and (d). (iii) Selecting a host cell, wherein the first circular DNA product comprising (a) and (b) is integrated into the target region of the genome via a first single crossover homologous recombination event between the flanking sequence of the target sequence and the target region of the host cell genome, and (iv) Select a Lactobacillus host cell in which (a) has been excised from the genome of the Lactobacillus host cell obtained in (iii) by a second homologous recombination event between the flanking sequence of the target sequence and the target region of the genome.

19. The method of claim 18 for introducing recombination between a circular DNA vector and a target region of a lactobacillus host cell genome, wherein the method produces a host cell with a mutation in the target gene or produces a host cell expressing a recombinant polypeptide.

20. The method of claim 19 for introducing recombination between a circular DNA vector and a target region of the Lactobacillus host cell genome, wherein the mutation is a loss-of-function mutation or a gain-of-function mutation.

21. The method of claim 19, wherein the recombinant polypeptide is a polypeptide selected from the group consisting of antibodies, enzymes, cytokines and hormones.

22. The method of claim 18, wherein the flanking sequence comprises a series of nucleosides in the range of 400 to 1500.

23. The method of claim 18, wherein the selection under (iii) uses an optional marker gene sequence (a) of the circular DNA vector.

24. The method of claim 18, wherein the selection under (iii) uses PCR and / or DNA sequencing.

25. The method of claim 18, wherein the host cell selected in (iv) is selected by anti-selection.

26. The method of claim 18, wherein the host cell selected under (iv) is negative for the selectable marker.

27. The method of claim 18, wherein the host cells selected in (iv) are selected using PCR.

28. The method of claim 18, wherein the product of (iv) is a host cell comprising a deletion of the target region, a partial deletion of the target region, a sequence insertion of the target region, a point mutation of the target region, or a sequence substitution of the target region.

29. The method of claim 18, wherein the lactobacillus host cell is selected from the group consisting of: Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus curvifolius, Lactobacillus janniae, Lactobacillus vaginalis, Lactobacillus inertia, Lactobacillus reuteri, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus curvifolius, Lactobacillus delbrueckii, and Lactobacillus johnsonii.

30. The method of claim 18, wherein the replicon comprises an origin of replication of Escherichia coli (E. coli).

31. The method of claim 18, wherein the replicon comprises repA encoding the regulatory protein RepA, repB encoding the replication protein RepFIB, or RepC encoding the replication initiation protein.

32. The method of claim 18, wherein the circular DNA vector is introduced by transformation.

33. The method of claim 18, wherein the target gene encodes a cell surface protein.

34. The method of claim 18, wherein the cell surface protein is a sorting enzyme-dependent protein (SDP) or an S-layer protein.

35. The method of claim 18, wherein the target gene encodes a cellular protein involved in the biosynthesis of cell surface molecules.

36. The method of claim 18, wherein the cell surface molecule is an extracellular polysaccharide (EPS).

37. The method of claim 18, wherein the target gene encodes a protein involved in bacterial adhesion, autoaggregation, and / or biofilm formation.

38. The method according to claim 18, wherein the host cell is *Lactobacillus gasseri* and the target gene is selected from the group consisting of N506_1709, N506_1778, N506_0396, N506_0397, N506_0398, N506_0399, N506_0400, N506_0401, N506_0402, N506_0403, N506_0404, N506_0405, N506_0406, N506_0407, N506_0408, N506_0409, N506_0410 and N506_0411.

39. The method of claim 18, wherein the host cell is Lactobacillus gasseri DSM 14869.

40. The use of the circular DNA vector according to claim 1 for introducing a gene sequence into the genome of a lactobacillus host cell.

41. The use according to claim 40, wherein the circular DNA vector introduces a deletion of the target region, a partial deletion of the target region, a sequence insertion in the target region, a point mutation in the target region, or a sequence substitution in the target region.

42. The use according to claim 40, wherein the gene sequence is expressed in the host cell.

43. The use according to claim 40, wherein the gene sequence blocks the expression of the endogenous host gene.

44. The use according to claim 40, wherein the gene sequence replaces the corresponding endogenous host gene.

45. The use according to claim 40, wherein the host cell is selected from the group consisting of: Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus curvifolius, Lactobacillus janniae, Lactobacillus vaginalis, Lactobacillus inertia, Lactobacillus reuteri, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus curvifolius, Lactobacillus delbrueckii, and Lactobacillus johnsonii.

46. ​​The use according to claim 40, wherein the host cell is *Lactobacillus gasseri* and the target gene is selected from the group consisting of N506_1709, N506_1778, N506_0396, N506_0397, N506_0398, N506_0399, N506_0400, N506_0401, N506_0402, N506_0403, N506_0404, N506_0405, N506_0406, N506_0407, N506_0408, N506_0409, N506_0410 and N506_0411.

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