Bacillus subtilis engineering bacteria, cascade catalysis construction method and application thereof

Abstract

The application discloses a bacillus subtilis engineering bacterium and a cascade catalysis construction method and application thereof, relates to the technical field of genetic engineering. The engineering bacterium is a bacillus subtilis engineering bacterium BS2024E1E2E3, has been preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC M 2024966 and the preservation date is May 16, 2024. The application realizes cascade catalysis agarose to produce active oligosaccharide and monosaccharide by integrating agarase into the bacillus subtilis genome for fusion expression. The biological activity of the active oligosaccharide and monosaccharide is used to treat enteritis. The engineering bacterium can effectively catalyze agarose to produce active oligosaccharide and monosaccharide, and the animal experiment proves that the engineering bacterium can effectively treat mouse enteritis, and therefore has good scientific significance and application value.

Description

Technical Field

[0001] This application relates to fields such as molecular biology, specifically to an engineered Bacillus subtilis strain and its cascade catalytic construction method and application. Background Technology

[0002] Inflammatory bowel disease (IBD) severely impacts human health, often causing gastrointestinal (GI) debilitating symptoms and even leading to cancer. While the exact pathogenesis of IBD remains unclear, it is associated with disruption of the gastrointestinal epithelial barrier and dysbiosis of the gut microbiota. Conventional symptom control relies on pharmacological interventions such as aminosalicylate, antibiotics, corticosteroids, and immunosuppressants. However, these treatments are limited by low efficacy and severe side effects, potentially causing headaches, seizures, neurological disorders, and nephrotoxicity. Therefore, developing a simple, safe, and effective IBD treatment strategy to induce and maintain effective remission long-term remains a challenging goal.

[0003] Dietary intervention has long been considered an acceptable and sustainable approach to controlling IBD symptoms by modulating the microbiome, tight junctions, and mucosal layer. However, traditional IBD dietary therapies are randomized, and the specific trigger foods vary greatly between individuals. Transforming regular foods into more specific and active substances is considered a promising alternative that can treat IBD more precisely and effectively. Agarose is a natural marine polysaccharide widely distributed in edible red algae, such as Gracilaria and Agaricus. Studies have shown that agarose degradation products, agarooligosaccharides, have significant anti-inflammatory, antioxidant, and prebiotic activities. Utilizing microorganisms to degrade agarose into active oligosaccharides in the gastrointestinal tract for the treatment and prevention of enteritis would be a convenient and effective dietary therapy model. However, the biggest bottleneck in achieving this goal is that the human body is usually unable to transform regular foods into specific active substances due to the lack of relevant microorganisms or functional enzymes in the gut. Summary of the Invention

[0004] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide an engineered Bacillus subtilis strain and its cascade catalytic construction method and application.

[0005] To address the aforementioned technical problems, this invention engineered the probiotic Bacillus subtilis to enable it to successfully exhibit three enzymes that degrade agarose, thereby completing a multi-enzyme cascade catalysis of agarose, obtaining active oligosaccharides, and effectively treating intestinal inflammation.

[0006] The technical solution of the present invention is as follows: The first aspect of the present invention provides an engineered Bacillus subtilis strain, wherein the engineered Bacillus subtilis strain ( Bacillus subtilisThe strain identified is Bacillus subtilis engineered strain BS2024E1E2E3, which has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2024966, on May 16, 2024. The deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0007] A second aspect of the present invention provides an agarase prepared from the engineered Bacillus subtilis strain.

[0008] Preferably, the agarase includes type I β-agarase, type II β-agarase, and α-neo-agarbiose hydrolase.

[0009] A third aspect of the present invention provides a gene encoding the agarase, the agarase comprising class I β-agarase, class II β-agarase, and α-neo-agarbiose hydrolase, wherein the nucleotide sequence of the gene encoding class I β-agarase comprises the sequence shown in SEQ ID NO.1, the nucleotide sequence of the gene encoding class II β-agarase comprises the sequence shown in SEQ ID NO.2, and the nucleotide sequence of the gene encoding α-neo-agarbiose hydrolase comprises the sequence shown in SEQ ID NO.3.

[0010] A fourth aspect of the present invention provides a recombinant vector containing the said gene.

[0011] The fifth aspect of the present invention provides a cascade catalytic construction method for the engineered Bacillus subtilis strain, the cascade catalytic construction method comprising: using Bacillus subtilis WTΔepsΔbslA as the starting strain, and transferring the recombinant vector into the starting strain.

[0012] A sixth aspect of the present invention provides an engineered biofilm comprising the engineered Bacillus subtilis bacteria and its secretions.

[0013] A seventh aspect of the present invention provides a living material for the preparation of active oligosaccharides and / or monosaccharides, the living material comprising the engineered Bacillus subtilis bacteria or the engineered biofilm.

[0014] The eighth aspect of the present invention provides an active oligosaccharide and / or monosaccharide, obtained by the degradation of agarose by the engineered Bacillus subtilis bacteria, the agarase, the engineered biofilm, or the living material.

[0015] The ninth aspect of the present invention provides the use of the engineered Bacillus subtilis strain, the agarase, the engineered biofilm, the living material, or the active oligosaccharide and / or monosaccharide in the preparation of a medicament for treating intestinal inflammation.

[0016] This invention has at least one of the following beneficial effects: This application obtains an engineered Bacillus subtilis strain by integrating agarose degradation to produce active oligosaccharide and monosaccharide elements. Bacillus subtilis BS2024E1E2E3, this engineered bacterium can produce three enzymes that degrade agarose: type I β-agarase, type II β-agarase, and α-neo-agarbiose hydrolase. Furthermore, this engineered bacterium, engineered biofilm, or the three enzymes produced can be used to develop a method for degrading agarose to produce active oligosaccharides and monosaccharides. Experiments have shown that degrading agarose to produce active oligosaccharides and monosaccharides enables tolerance to the complex environment of the gastrointestinal tract and treatment of intestinal inflammation. Therefore, this invention provides engineered bacteria, engineered biofilms, and biofilm living materials for achieving tolerance to the complex environment of the gastrointestinal tract and treatment of intestinal inflammation, thus providing a simple, effective, and sustainable dietary therapy method for enteritis. Attached Figure Description

[0017] Figure 1 This is a characterization and verification diagram of the engineered biofilm in Example 1.

[0018] Figure 2 The image shows a thin-layer chromatography (TLC) image of the engineered biofilm in Example 2, illustrating the production of active oligosaccharides and monosaccharides from agarose by different agarases.

[0019] Figure 3 Example 3 shows the efficacy of engineered Bacillus subtilis biofilms (E1E2E3) and agarose in treating enteritis in mice. In the figure, A is the modeling of IBD mice and the treatment plan of engineered strains; B is the change in body weight of mice in different treatment groups; C is the disease index of mice in different treatment groups; D is the colon tissue of mice in different treatment groups; E is the comparison of colon length of mice in different treatment groups; F is the HE staining of colon tissue sections of mice in different treatment groups; and G is the comparison of myeloperoxidase (MPO) activity of colon tissue of mice in different treatment groups.

[0020] Figure 4 This is a biosafety evaluation diagram of the engineered strain in Example 4. In the diagram, A-D represent the number of viable bacteria in different parts of the gastrointestinal tract after oral administration of the engineered strain at different growth stages for different durations. E represents the statistical analysis of blood routine and liver and kidney function in mice after oral administration of the engineered strain and low-melting-point agarose. F represents the HE staining diagram of visceral sections after oral administration of the engineered strain and low-melting-point agarose. Detailed Implementation

[0021] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0022] As used in this application, "nucleotide sequence" means a sequence of nucleotides from the 5' to 3' ends of a nucleic acid molecule and includes DNA or RNA molecules, including cDNA, DNA fragments or portions, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA, and antisense RNA, any of which may be single-stranded or double-stranded.

[0023] As used in this application, "promoter" refers to a nucleic acid region located upstream of the translation start codon and involved in the recognition and binding of RNA polymerase and other transcription proteins.

[0024] As used in this application, "regulator" refers to the base sequence of a functional promoter and any associated transcriptional element (e.g., enhancer, CCAAT box, TATA box, SPI part, etc.).

[0025] As used in this application, "plasmid" refers to an autonomous circular DNA molecule capable of replicating in a cell.

[0026] As used in this application, “expression” includes any step involved in peptide production, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0027] This application provides an engineered bacterium whose genome includes integrated expression of agarose degradation elements and biofilm component elements.

[0028] The prevalence of inflammatory bowel disease (IBD) severely impacts human health, often causing gastrointestinal (GI) debilitating symptoms and even leading to cancer. Probiotic therapy has shown great potential in this field due to its persistence and site specificity; however, most probiotics cannot withstand the complex environment of the gastrointestinal tract. To address these issues, this application aims to construct engineered bacteria using safe and non-toxic Bacillus subtilis to produce a biofilm capable of resisting the complex environment of the gastrointestinal tract while successfully demonstrating class I and II β-agarases and α-neo-agarbiose hydrolases, producing active oligosaccharides and monosaccharides to treat intestinal inflammation.

[0029] Optionally, in all embodiments of this application, the engineered bacteria are selected from Bacillus subtilis. Preferably, the engineered bacteria are selected from Bacillus subtilis WTΔepsΔbslA. Bacillus subtilis WTΔepsΔbslA is a Bacillus subtilis mutant strain obtained by gene editing technology, by knocking out the eps gene cluster and the bslA gene from a wild-type Bacillus subtilis WT strain. Specifically, homologous recombination or gene editing technologies such as CRISPR-Cas9 can be used to precisely knock out the eps gene cluster and the bslA gene. Since gene editing technology is well known to those skilled in the art, it will not be described in detail in this embodiment.

[0030] Optionally, in all embodiments of this application, the agarose degradation element includes three types of agarase elements: type I β-agarase, type II β-agarase, and α-neo-agarbiose hydrolase. The nucleotide sequence of type I β-agarase includes the sequence shown in SEQ ID NO.1, the nucleotide sequence of type II β-agarase includes the sequence shown in SEQ ID NO.2, and the nucleotide sequence of α-neo-agarbiose hydrolase includes the sequence shown in SEQ ID NO.3.

[0031] SEQ ID NO.1:

[0032] SEQ ID NO.2:

[0033] SEQ ID NO.3:

[0034] Specifically, the engineered bacteria in this application are Bacillus subtilis engineered bacteria (Bacillus subtilis engineered bacteria) Bacillus subtilis Lot BS2024E1E2E3 has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2024966, on May 16, 2024. The deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province. This application also provides an engineered biofilm, which includes the engineered bacteria and their secretions.

[0035] This application also provides a living material for the preparation of active oligosaccharides and monosaccharides, the living material comprising the engineered bacteria or engineered biofilm.

[0036] This application also provides the use of the engineered bacteria, living materials, or engineered biofilms in the preparation of drugs for treating intestinal inflammation.

[0037] Specifically, the engineered bacteria, living materials, or engineered biofilms can degrade agarose and produce active oligosaccharides and monosaccharides for the treatment of intestinal inflammation.

[0038] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following specific embodiments.

[0039] Example 1: Construction of Bacillus subtilis multi-component agarase display engineered strain BS2024E1E2E3 (1) Strain WTΔ eps Δ bslA Δ amyE Construction of ::E1E3 Using pDG1730 plasmid as a backbone, through Bam HI and Eco Linear plasmids were obtained by double digestion with RI. bslA-E1-his was synthesized by Genewiz Biotechnology Co., Ltd., and used as a template for PCR amplification; P bslA The gene of Bacillus subtilis was used as a template and obtained by PCR. The enzyme-digested linear plasmid was then combined with the PCR fragments bslA-E1-his and P... bslA Plasmid pDG-P was obtained by ligation through seamless cloning. bslA -bslA-E1-his. Using this plasmid as a backbone, through... Eco RV and Dra Double digestion with enzyme III yielded linear plasmids. E3-his was synthesized by Genewiz Biotechnology Co., Ltd., and P-SpoIIIJ was obtained by PCR using the Bacillus subtilis genome as a template. These two fragments were seamlessly cloned and ligated with the plasmids to obtain plasmid pDG-P. bslA-bslA-E1-his-P-SpoIIIJ-E3-his.

[0040] plasmid pDG-P bslA -bslA-E1-his-P-SpoIIIJ-E3-his was transformed into Bacillus subtilis WTΔ eps Δ bslA In the process, strain WTΔ was obtained. eps Δ bslA Δ amyE ::E1E3.

[0041] (2) BS E1E2E3 Construction of strains The extracellular polysaccharides of Bacillus subtilis can regulate the intestinal immune system and maintain intestinal health. Therefore, [the following is a list of ingredients / products]. tasA-E2 In place, type in tasAsinR Site, simultaneously knocked out sinR Genes, to relieve its inhibition of extracellular polysaccharides.

[0042] Using pJOE8999 as the plasmid backbone, searched the NCBI website. tasAsinR The gene and its upstream and downstream 1000 bp sequences will tasAsinR The gene was copied and pasted to the website to design the sgRNA. After design, it was submitted to Qingke Biotechnology Co., Ltd. for synthesis. The synthesized primers were annealed to form double strands. pJOE8999 was then... Bsa After digestion with enzyme I, the plasmid backbone was obtained by gel extraction. The annealed double-stranded sgRNA was ligated to the digested pJOE8999 plasmid using T4 ligase, followed by sequencing to obtain pJOE8999-TS. tasAsinR The gene and its upstream and downstream 1000 bp sequences are used as homologous arms, with the upstream 1000 bp designated as "up" and the downstream 1000 bp as "down". Gene fragments "up", "E2", and "down" are obtained using PCR, and seamless cloning technology is then used... Sfi The three gene fragments were cloned into pJOE8999-TS by restriction enzyme I, resulting in the plasmid pJOE8999-TS-tasA-E2. The plasmid pJOE8999-TS-tasA-E2 was then transformed into WTΔ. bslA Δ amyE ::E1E3, and will tasA-E2 type tasAsinR , obtain WTΔ bslA Δ amyE ::E1E3Δ tasAsinR :: tasA-E2 (BS) E1E2E3 The engineered strain BS2024E1E2E3 was constructed. Genome PCR sequencing confirmed successful integration of the target fragment.

[0043] This embodiment also constructed negative controls for biofilm-deficient strains, including Control, E1 (BslA-E1), E2 (TasA-E2), E3 (SpoIIIJ-E3), E1E2 (BslA-E1-TasA-E2), and E1E3 (BslA-E1-SpoIIIJ-E3). Each group was constructed according to the following method: With WTΔ eps Δ bslA Δ tasA Δ sinR As a negative control strain, plasmid pDG-P was used. bslA -bslA-E1-his、pDG-P tasA -tasA-E2, pDG-P-SpoIIIJ-E3-his, pDG-P bslA -bslA-E1-his-P tasA -tasA-E2、pDG-P bslA -bslA-E1-his-P-SpoIIIJ-E3-his is converted to WTΔ eps Δ bslA Δ tasA Δ sinR Strains were obtained that expressed BslA-E1, TasA-E2, SpoIIIJ-E3 and BslA-E1-TasA-E2, BslA-E1-SpoIIIJ-E3, respectively.

[0044] To verify that the engineered strain BS2024E1E2E3 of this embodiment successfully displayed agarase on a biofilm, the present invention used transmission electron microscopy (TEM) and immunofluorescence (IF) to observe the formation of hydrophobic proteins of engineered strain BslA-E1 and fibers of TasA-E2.

[0045] Figure 1 To confirm the successful display of agarose-degrading enzymes E1E2E3 on biofilms using transmission electron microscopy (TEM) and immunofluorescence (IF), an engineered biofilm was successfully constructed. Figure 1 The first four images are TEM images. Figure 1 The last two images are IF, TEM scale bar: 0.5 μm, IF scale bar: 2 μm. See details below. Figure 1As shown, no significant formation of hydrophobic proteins and fibers was observed in the negative control (Control) of biofilm-deficient strains, while a large amount of hydrophobic proteins and fibers were formed around the engineered strains that successfully expressed the BslA-E1 and TasA-E2 fusion proteins. Immunofluorescence revealed significant fluorescence in the engineered strains expressing the SpoIIIJ-E3 fusion protein, while the negative control (Control) strains of biofilm-deficient strains showed no significant fluorescence. Therefore, it is demonstrated that the biofilm of the engineered strains successfully displayed the expression of three agarases E1, E2, and E3.

[0046] Example 2: Production of bioactive oligosaccharides and monosaccharides by engineered strains degrading agarose. To verify the successful display of agarase on a multi-component biofilm and its ability to cascade the production of active oligosaccharides and monosaccharides from agarose, this example will demonstrate engineered strains obtained in Example 1 with a single enzyme (E1), two enzymes (E1 and E2, E1 and E3), and three enzymes (E1, E2, and E3) at a ratio of 10... 8 -10 9 CFUs were inoculated in MSgg medium containing 0.1% (wt) low-melting-point agarose and cultured statically. Samples were taken every 12 hours to detect the reducing sugar content (DNS method). After 60 hours of enzymatic hydrolysis, samples were taken for thin-layer chromatography to investigate the degree of polymerization of the hydrolysis products.

[0047] like Figure 2 As shown, with the continuous formation of the biofilm, enzymatic hydrolysis products are gradually generated, and the content of reducing sugars continuously increases. TLC data show that the enzymatic hydrolysis product of BslA-E1 is mainly neoagartetraose. TasA-E2 and BslA-E1 form a cascade catalysis to enzymatically hydrolyze low-melting-point agarose into neoagarbiose. SpoIIIJ-E3 further enzymatically hydrolyzes the products of E1E2 to produce 3,6-endoether-L-galactose.

[0048] The above experimental results demonstrate that E1 displayed on BslA and E2 displayed on TasA both possess the ability to catalyze the degradation of agarose to produce active oligosaccharides, while E3 displayed on the cell surface can further degrade the enzymatic hydrolysis products (tetrasaccharides and disaccharides) of E1 and E2 into monosaccharides. This indicates that rationally designed engineered... B. subtilis Biofilm materials can be used for multi-enzyme display, realizing the potential of multi-enzyme synergy and cascade catalysis applications.

[0049] Example 3: Engineered strains catalyze the production of active oligosaccharides and monosaccharides from agarose for the treatment of enteritis. Seven-week-old female C57BL / 6 mice were randomly divided into five groups of three.

[0050] Group 1 (DSS group): Healthy mice were given sterile H2O orally daily without any other treatment. The remaining 4 groups of mice were given 3% DSS freely for 5-7 days. The mice were weighed daily, and the shape of their feces and the presence of blood in their stool were recorded and scored. Once the mouse colitis model was successfully established, the 3% DSS was replaced with sterile H2O.

[0051] Group 2 (PBS group): Model mice were administered PBS by gavage.

[0052] Group 3 (DSS+Bs) E1E2E3 Group, abbreviated as Bs E1E2E3 ): Model mice, treated on day 1 with engineered strain BS2024E1E2E3 spores (1 × 10⁻⁶). 8 Mice were administered CFUs by gavage, followed by PBS by gavage for the next 4 days.

[0053] Group 4 (DSS+Aga group, abbreviated as Aga): Model mice were administered low-melting-point agar (1×10⁻⁶) by gavage daily. -5 (mg / kg).

[0054] Group 5 (DSS+BsE@A group, abbreviated as BsE@A): Model mice, treated on day 1 with engineered strain BS2024E1E2E3 spores (1 × 10⁻⁶). 8 CFUs) and low melting point agar (1×10⁻⁶) -5 Administered via gavage at mg / kg, followed by 4 days of treatment with low-melting-point agar (1×10 mg / kg). -5 (mg / kg) administered by gavage.

[0055] Group 6 (DSS+5-ASA group, abbreviated as 5-ASA): model mice were administered 5-aminosalicylic acid by gavage daily.

[0056] On day 5 of treatment, feces were collected from the mice, and the mice were euthanized. The mice were dissected, and the colon was removed to measure its length and morphology. After measurement, approximately 1 cm of the colon was cut off and immersed in 4% paraformaldehyde for HE staining. The remaining colon was collected in sterile centrifuge tubes and stored at -80 °C for enzyme-linked immunosorbent assay (ELISA) analysis of colitis-related markers.

[0057] like Figure 3As shown, after induction with 3% DSS, mice in the DSS group exhibited decreased activity, loose stools, bloody stools, and a 26% decrease in body weight. Dissection revealed a significant shortening of the colon in the DSS group mice compared to healthy mice. H&E staining of colonic tissue showed obvious colonic ulcers, mucosal edema, goblet cell loss, crypt swelling and destruction, and varying degrees of inflammatory cell infiltration and epithelial cell damage in the mucosa and submucosa. Myeloperoxidase activity in colonic tissue was significantly increased in the DSS group mice; these experimental results indicate successful establishment of a mouse colitis model. The engineered strain (DSS+Bs) was administered alone. E1E2E3 In the control group (DSS+Aga group), mice fed with either engineered bacterial strains or 0.1% low-melting-point agarose showed a slight increase in body weight, but this was still lower than in healthy mice. The colon length was longer than in the DSS group but shorter than in healthy mice. HE staining of the colon tissue showed no significant relief of colitis, and the histopathological score was high. Myeloperoxidase activity and inflammatory factors remained higher than in healthy mice. However, in the groups simultaneously administered engineered bacterial strains and low-melting-point agarose, especially the DSS+BsE@A group, mouse body weight significantly increased, and activity levels rose. The colon length returned to the level of healthy mice, and HE staining of the colon tissue showed a lower histopathological score, close to the control group (PBS group). The epithelium was intact, the finger crypt structure was regular, and the percentage of inflammatory cells was low. This experiment demonstrates that feeding engineered bacterial strains plus low-melting-point agarose can significantly promote the repair of colon tissue in mice, effectively treating enteritis.

[0058] Example 4: Biosafety Evaluation of Engineered Strains 1. Validation of strain tolerance After culturing the engineered strain BS2024E1E2E3, planktonic cells, biofilms, and spores of the engineered strain BS2024E1E2E3 were obtained, respectively. The planktonic cells, biofilms, and spores of the engineered strain BS2024E1E2E3 were then cultured at a ratio of 1×10⁻⁶. 8 CFUs were fed to healthy C57BL / 6 mice. The mice were euthanized 24 h, 48 h, and 72 h after gavage, and their stomachs, intestines, colons, and cecums were collected, homogenized, and serially diluted. 100 μL of each dilution was spread onto solid LB agar plates containing 100 μg / mL spectinomycin and incubated for 12 h. The number of colonies on each plate was then counted.

[0059] The results are as follows Figure 4 A- Figure 4As shown in D, it can be seen that the engineered strain was almost undetectable in the gastrointestinal tract of mice administered the planktonic strain by gavage. However, the target strain was detectable in the gastrointestinal tract of mice administered both biofilm and spores by gavage. The spores were almost unaffected by the complex gastric environment; even 72 hours after oral administration, the engineered strain (~10⁻⁶) was still detectable in the gastrointestinal tract. 4 The presence of CFUs indicates that the spores of the engineered strain BS2024E1E2E3 can withstand the complex environment of the gastrointestinal tract.

[0060] 2. Biosafety evaluation of orally administered engineered strains Healthy C57BL / 6 mice were randomly divided into two groups.

[0061] Blank control group: Healthy mice, no treatment.

[0062] Experimental group (BsE@A): Healthy mice were orally administered low-melting-point agarose (1×10⁻⁶ by mass) on day 1. -5 1×10 mg / kg resuspended 8 CFU engineered strain BS2024E1E2E3 (final volume 200 μL), followed by oral administration of 200 μL of low-melting-point agarose (1×10⁻⁶). -5 mg / kg). On day 5 post-treatment, mice in both the control and experimental groups were euthanized, and major organ (heart, liver, spleen, lungs, and kidneys) and blood samples were collected. Histological changes in the organs were analyzed by H&E staining, and whole blood samples were used for hematological analysis (Sysmex XE-2100, Kobe, Japan). Analyzed blood parameters included white blood cells (WBC), red blood cells (RBC), and hemoglobin (HGB). Liver function indicators (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) and kidney function indicators (blood urea nitrogen (BUN) levels) were analyzed using a BS-2000 m automated biochemical analyzer.

[0063] like Figure 4 As shown in E, in this experiment, serum biochemical analysis and whole blood tests were performed after multiple oral administrations of the engineered strain. As expected, the blood cell parameters of mice treated with the engineered strain, including white blood cells, red blood cells, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, hemoglobin, and platelets, were similar to those of healthy mice. Liver and kidney function parameters, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin (ALB), were also within the normal range. Furthermore, compared to healthy mice, the mice treated with the engineered strain showed almost no difference in body weight and histological sections of major organs. Figure 4 (F). All these results indicate that oral administration of the engineered strain did not cause any adverse side effects in mice.

[0064] This application demonstrates the cascade catalytic degradation of agarose to produce bioactive oligosaccharides by displaying agarase on multiple components of a Bacillus subtilis biofilm. When the engineered strain and agarose were administered via gavage to DSS-induced enteritis mice, the engineered bacteria effectively improved the mice's enteritis by degrading the bioactive oligosaccharides and monosaccharides produced from the agarose, and no detectable toxic side effects were observed. This opens up a new avenue for dietary therapy.

[0065] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An engineered Bacillus subtilis strain, characterized in that, The Bacillus subtilis engineered strain ( Bacillus subtilis The strain identified is Bacillus subtilis engineered strain BS2024E1E2E3, which has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2024966, on May 16, 2024. The deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. An agarase, characterized in that, The agarase was prepared from the engineered Bacillus subtilis strain described in claim 1.

3. The agarase according to claim 2, characterized in that, The agarases include type I β-agarase, type II β-agarase, and α-neo-agarbiose hydrolase.

4. A gene encoding the agarase according to any one of claims 2 to 3, characterized in that, The agarases include type I β-agarase, type II β-agarase, and α-neo-agarbiose hydrolase. The nucleotide sequence of the gene encoding type I β-agarase includes the sequence shown in SEQ ID NO.1, the nucleotide sequence of the gene encoding type II β-agarase includes the sequence shown in SEQ ID NO.2, and the nucleotide sequence of the gene encoding α-neo-agarbiose hydrolase includes the sequence shown in SEQ ID NO.

3.

5. A recombinant vector, characterized in that, It contains the gene described in claim 4.

6. A cascade catalytic construction method for the engineered Bacillus subtilis strain as described in claim 1, characterized in that, The cascade catalytic construction method includes: using Bacillus subtilis WTΔepsΔbslA as the starting strain, and transferring the recombinant vector of claim 5 into the starting strain.

7. An engineered biofilm, characterized in that, The engineered biofilm comprises the engineered Bacillus subtilis strain of claim 1 and its secretions.

8. A living material for the preparation of active oligosaccharides and / or monosaccharides, characterized in that, The living material includes the engineered Bacillus subtilis strain of claim 1 or the engineered biofilm of claim 7.

9. An active oligosaccharide and / or monosaccharide, characterized in that, It is produced by the degradation of agarose by the engineered Bacillus subtilis strain of claim 1, the agarase of claim 2, the engineered biofilm of claim 7, or the living material of claim 8.

10. Use of the engineered Bacillus subtilis strain of claim 1, or the agarase of claim 2, or the engineered biofilm of claim 7, or the living material of claim 8, or the active oligosaccharide and / or monosaccharide of claim 9 in the preparation of a medicament for treating intestinal inflammation.

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