An adhesive engineered yeast probiotic preparation and a preparation method and application thereof

Abstract

The present application belongs to the field of microbial technology and medicine, and particularly relates to an adhesive engineered yeast probiotic preparation and a preparation method and application thereof. The present application uses synthetic biology technology to construct an engineered yeast strain capable of efficiently expressing canthaxanthin. On this basis, an adhesive engineered yeast probiotic MBP_Sc22 is prepared through norepinephrine self-polymerization, which improves the survival rate and retention capacity of the engineered yeast in the gastrointestinal tract without impairing the ability of the engineered yeast to target macrophages. The adhesive engineered yeast probiotic preparation expresses canthaxanthin, removes active oxygen at the inflammation site, down-regulates the inflammation level, increases the intestinal microbial abundance and diversity, optimizes the intestinal microbial structure and restores the intestinal microbial homeostasis, thereby achieving the purpose of treating colitis.

Description

Technical Field

[0001] This invention belongs to the fields of microbial technology and medicine, specifically relating to an adhesive engineered yeast probiotic preparation, its preparation method, and its application. Background Technology

[0002] The gut microbiota, composed of a large number of microorganisms, is considered the second genome of humans. Probiotics, through spatial competition, biological antagonism, and the secretion of metabolites, provide the host with various beneficial effects, such as immune regulation, intestinal barrier strengthening, and promotion of nutrient metabolism. With the development of synthetic biology, engineered bacteria therapy, which involves the targeted modification of traditional probiotics to obtain specific functions, has shown significant potential in the treatment of various diseases, including diabetes, phenylketonuria, cancer, obesity, and inflammatory bowel disease (IBD).

[0003] Inflammatory bowel disease (IBD) is a group of chronic inflammatory diseases affecting both the small and large intestines. Based on the affected sites, it is mainly divided into ulcerative colitis (UC) and Crohn's disease (CD). Major clinical manifestations include abdominal pain, diarrhea, and rectal bleeding, severely impacting patients' quality of life. However, the complex pathogenesis of IBD makes current treatments insufficient to achieve ideal therapeutic effects. Most commonly used drugs for IBD (such as aminosalicylic acid derivatives, glucocorticoids, and immunosuppressants) fail to address the core pathogenic mechanisms of IBD, such as oxidative stress, intestinal mucosal damage, immune abnormalities, and gut microbiota dysbiosis. Furthermore, long-term use of these drugs can easily lead to drug resistance or systemic side effects. Therefore, a safe and effective treatment strategy to address the root causes of IBD is urgently needed.

[0004] Engineered probiotics can regulate intestinal inflammation by modulating the gut microbiota, precisely delivering therapeutic factors, and interacting with the host immune system, making them a promising new drug strategy in the treatment of IBD in recent years. Yan et al. engineered *E. coli* Nissle 1917 to express 3-hydroxybutyric acid (3HB), enabling it to colonize the mouse intestine and continuously produce 3HB, thereby targeting and alleviating colitis in mice (Yan et al., 2021). Furthermore, Scott et al. developed a self-regulating engineered yeast probiotic that, through directed evolution, enhances the sensitivity of the P2Y2 receptor to inflammatory signals and couples it with adenosine triphosphate diphosphatase to clear pro-inflammatory molecules, significantly alleviating intestinal damage in UC mice (Scott et al., 2021). Notably, *Saccharomyces cerevisiae* (Saccharomyces cerevisiae)... Saccharomyces cerevisiaeAs a GRAS (Generally Recognized As Safe) eukaryotic host, yeast possesses characteristics such as rapid growth and reproduction, short metabolic cycle, and clear genetic background, making it one of the most widely used microorganisms in the biotechnology field, extensively applied in fermented foods, feed, and bioproducts industries. Furthermore, β-1,3-D-glucan in the yeast cell wall possesses unique immunomodulatory activity and serves as a "recognition signal" mediating delivery to macrophages (Dectin-1 receptor), making it an ideal choice for developing oral engineered probiotics, especially providing new research ideas for targeted intervention in UC (ulcerative colitis). Meanwhile, cantharidin, as an active molecule with both antioxidant and immunomodulatory functions, has shown outstanding efficacy in restoring intestinal mucosal barrier function and microbial homeostasis in UC mice (Chen et al., 2025). Therefore, developing engineered yeast strains that efficiently express cantharidin is a highly promising biotherapy for UC treatment. However, orally administered engineered probiotics often face the harsh environment of the gastrointestinal tract, including gastric acid, digestive enzymes, and bile salts, as well as the "colonization resistance" of resident gut microbiota, resulting in limited survival and retention time in the gut and severely weakening therapeutic effects. Therefore, developing a biofilm-coated, protected oral engineered yeast probiotic treatment strategy is of significant practical importance for improving the efficacy of engineered probiotics in treating ulcerative colitis (UC). Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of this invention is to provide a technical solution for an adhesive engineered yeast probiotic preparation, its preparation method and application.

[0006] The first aspect of this invention provides a method for preparing an adhesive engineered yeast probiotic preparation, the specific steps of which include: adding norepinephrine to sterile PBS to prepare a solution, and then adding Saccharomyces cerevisiae (Saccharomyces cerevisiae)... Saccharomyces cerevisiae MBP_Sc22 was resuspended in norepinephrine solution, shaken and incubated at low temperature, washed and resuspended with physiological saline to obtain the final product. The preservation number of the brewing yeast is: CGMCC No. 35982.

[0007] Furthermore, the concentration of norepinephrine is 0.5-1 mg / mL, and the concentration of the final product is 3.5 × 10⁻⁶ mg / mL. 8 -4.5×10 8 CFU / mL.

[0008] Furthermore, the brewing yeast ( Saccharomyces cerevisiae The concentration of MBP_Sc22 was 3.5 × 10⁻⁶. 8 -4.5×10 8 CFU / mL.

[0009] The second aspect of the present invention provides an adhesive engineered yeast probiotic preparation obtained by the above method.

[0010] The third aspect of this invention provides the application of the above-mentioned adhesive engineered yeast probiotic preparation in the preparation of products for treating colitis.

[0011] The fourth aspect of this invention provides the application of the above-mentioned adhesive engineered yeast probiotic preparation in the preparation of products for regulating the intestinal flora of colitis.

[0012] The fifth aspect of the present invention provides a product for treating colitis, comprising the above-mentioned adhesive engineered yeast probiotic preparation.

[0013] The beneficial effects of this invention include:

[0014] This invention utilizes synthetic biology techniques to construct an engineered yeast strain that efficiently expresses canthaxanthin. Based on this, an adhesive engineered yeast probiotic was prepared through norepinephrine self-polymerization. This improved the survival rate and retention capacity of the engineered yeast in the gastrointestinal tract without impairing its ability to target macrophages. The described adhesive engineered probiotic preparation, by expressing canthaxanthin, selectively scavenges reactive oxygen species at sites of inflammation, downregulates inflammation levels, and simultaneously increases the abundance and diversity of gut microbiota, optimizes gut microbiota structure, and restores gut microbiota homeostasis, thereby achieving the therapeutic effect on colitis. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of gene elements for gene fragment 1; where TRP1 LHA and TRP1 RHA at both ends represent the upstream and downstream homologous sequences of the yeast TRP1 site, respectively.

[0016] Figure 2 This is a schematic diagram of gene elements for gene fragment 2; where ypl062w LHA and ypl062w RHA at both ends represent the upstream and downstream homologous sequences of the yeast ypl062w site, respectively.

[0017] Figure 3 This is a schematic diagram of gene elements for gene fragment 3; where gal7 LHA and gal1 RHA at both ends represent the upstream homologous sequence of the yeast gal7 site and the downstream homologous sequence of the gal1 site, respectively.

[0018] Figure 4 This is a schematic diagram of gene elements for gene fragment 4; where LEU2 LHA and LEU2 RHA at both ends represent the upstream and downstream homologous sequences of the yeast LEU2 site, respectively.

[0019] Figure 5 This is a schematic diagram of gene elements for gene fragment 5; where gal80 LHA and gal80 RHA at both ends represent the upstream and downstream homologous sequences of the yeast gal80 site, respectively.

[0020] Figure 6 The image shows the pJET1.2 plasmid.

[0021] Figure 7 Image of plasmid pRS405;

[0022] Figure 8 Characterization of CX-Sc; (A) CX-Sc fermentation chromatogram after 36 h; (B) HPLC peak chromatogram of cantharidin;

[0023] Figure 9 Preparation of NE-CX-Sc; (A) Physical image of NE-CX-Sc; (B) Quantitative analysis of norepinephrine on the surface of NE-CX-Sc; (C) Effect of NE coating on CX-Sc activity; (D) Zeta potential of NE-CX-Sc;

[0024] Figure 10 Characterization of the ultrastructure of NE-CX-Sc;

[0025] Figure 11 Survival of NE-CX-Sc exposed to gastrointestinal digestive fluids; (A) artificial gastric juice (SGF); (B) artificial intestinal juice (SIF);

[0026] Figure 12 The uptake of NE-CX-Sc by macrophages; Scale bar: 20 μm;

[0027] Figure 13 The adhesion ability of NE-CX-Sc to colonic epithelial cells; Scale bar: 50 μm;

[0028] Figure 14 (A) Fluorescence image of mouse gastrointestinal tract; (B) Quantitative analysis of fluorescence intensity in gastrointestinal tract.

[0029] Figure 15 The therapeutic effect of NE-CX-Sc on DSS-induced colitis in mice; (A) Modeling protocol; (B) Perianal images and fecal occult blood tests of mice; (C) Mouse weight change rate; (D) Disease activity index; (E) Quantitative map of colon length; (F) Images of mouse colon; (G) Colon H&E staining images: ae scale bar: 200 μm; fj scale bar: 50 μm;

[0030] Figure 16 Effect of NE-CX-Sc on ROS levels in colon tissue of UC mice; (A) Image of colon stained with DHE; scale bar: 200 μm; (B) Quantification of DHE relative fluorescence intensity;

[0031] Figure 17The effects of NE-CX-Sc on inflammatory factors in the intestinal tissue of UC mice; (A) tumor necrosis factor-α; (B) interleukin-1β; (C) interleukin-10; (D) interleukin-6;

[0032] Figure 18 For the biosafety evaluation of NE-CX-Sc: (A) alanine aminotransferase (ALT); (B) aspartate aminotransferase (AST); (C) creatinine; (D) blood urea nitrogen;

[0033] Figure 19 The effects of NE-CX-Sc on the gut microbiota of UC mice; (A) Chao index; (B) Shannon-Wiener index; (C) relative abundance of Bacteroides; (D) relative abundance of Akkermansia; (E) relative abundance of Shigella. Detailed Implementation

[0034] To fully disclose the adhesive engineered yeast probiotic of the present invention and its preparation and application, the following examples are provided, but this does not imply any limitation on the present invention.

[0035] Some of the plasmid vectors and strains involved in the embodiments of this invention are commercially available. For example, the pJET1.2 plasmid vector was purchased from Thermo Scientific's CloneJET PCR Cloning Kit, #K1231 (see plasmid map). Figure 6 ); plasmid pRS405 (plasmid map see...) Figure 7 The *Saccharomyces cerevisiae* strain CEN.PK2-1D was purchased from the EUROSCARF of Scientific Research and Development GmbH, Germany; *Saccharomyces cerevisiae* BY4742 was purchased from the National Center for Type Culture Collection.

[0036] The gene elements used in constructing the recombinant yeast strain of this invention, such as amino acid markers, tags, endogenous genes, and exogenous genes, are all well-known in the art, and their specific sequences are known to those skilled in the art. To facilitate understanding of this invention, the gene elements in each gene fragment are described below:

[0037] Gene fragment 1 (SEQ ID NO. 1) containing the CarB and CarRP genes from *Mucor truncatula*: 1-631bp is the 631bp homologous sequence upstream of the TRP1 site; 632-886bp is the CYC1 terminator sequence; 887-2626bp is the CarB gene from *Mucor truncatula*; 2627-3294bp is the GAL10-GAL1 bidirectional promoter sequence; 3295-5139bp is the CarRP gene from *Mucor truncatula*; 5140-5414bp is the PGK1 terminator sequence; 5415-6147bp is the 733bp homologous sequence downstream of the TRP1 site.

[0038] Gene fragment 2 (SEQ ID NO. 2) containing the CarB and CarRP genes from *Mucor truncatula*: 1-394bp is the 394bp homologous sequence upstream of the ypl062w site; 395-1951bp is the DR-URA3-DR nutrient tag sequence; 1952-2206bp is the CYC1 terminator sequence; 2207-3946bp is the CarB gene from *Mucor truncatula*; 3947-4614bp is the GAL10-GAL1 bidirectional promoter sequence; 4615-6459bp is the CarRP gene from *Mucor truncatula*; 6460-6734bp is the PGK1 terminator sequence; 6735-7051bp is the 317bp homologous sequence downstream of the ypl062w site.

[0039] Gene fragment 3 (SEQ ID NO. 3) containing the CrtE gene from Archaeocystis scintillans: 1-426bp is a 426bp homologous sequence upstream of the gal7 site; 427-1983bp is the DR-URA3-DR nutrient tag sequence; 1984-3330bp is the ERG10 gene and its terminator sequence; 3331-3836bp is the GAL7 promoter sequence; 3837-4123bp is the ACT1 terminator sequence; 4124-5632bp is the truncated HMG-CoA reductase gene tHMGR1; 5633-6300bp is the GAL10-GAL1 bidirectional promoter sequence; 6301-7254bp is the CrtE gene from Archaeocystis scintillans; 7255-7654bp is the GPM1 terminator sequence; 7655-7888bp is a 234bp homologous sequence downstream of the gal1 site.

[0040] Gene fragment 4 (SEQ ID NO. 4) containing the CrtW gene from Chlamydomonas reinhardtii: 1-561bp is a 561bp homologous sequence upstream of the LEU2 site; 562-1656bp is the LEU2 marker; 1657-2056bp is the TDH2 terminator sequence; 2057-2513bp is the GAL1 promoter sequence; 2514-3848bp is the CrtW gene from Chlamydomonas reinhardtii; 3849-4067bp is the SpO1 terminator sequence; 4068-4651bp is a 584bp homologous sequence downstream of the LEU2 site.

[0041] Gene fragment 5 (SEQ ID NO. 5) containing the CrtW gene from Chlamydomonas reinhardtii: 1-362bp is a 362bp homologous sequence upstream of the gal80 site; 363-1919bp is the DR-URA3-DR nutrient tag sequence; 1920-2691bp is the GAL1 promoter sequence; 2692-4026bp is the CrtW gene from Chlamydomonas reinhardtii; 4027-4245bp is the SpO1 terminator sequence; 4246-4699bp is a 454bp homologous sequence downstream of the gal80 site.

[0042] Once the specific sequences of the aforementioned gene elements are known, those skilled in the art can perform amplification and OE-PCR assembly according to conventional primer design principles. Furthermore, the SD medium used in this invention is a commonly used medium in the field of yeast screening. Target strains are screened by intentionally removing one or more components from the basic medium based on the specific gene defects present in the yeast.

[0043] Example 1: Construction of the cantharidin-producing engineered yeast MBP_Sc22

[0044] I. Experimental Materials

[0045] (1) Culture medium

[0046] YPD liquid culture medium: glucose 25 g / L, peptone 20 g / L, yeast extract 10 g / L.

[0047] II. Experimental Methods

[0048] 1. MBP_Sc22 Construction Method

[0049] (1) Explanation of the source of gene elements

[0050] The exogenous genes involved in this invention include the geranylide pyrophosphate synthase gene CrtE, the bifunctional enzyme gene CarRP (phytoene synthase / lycopene cyclase), the phytoene dehydrogenase gene CarB, and the β-carotene ketolase gene CrtW. Among them, CrtE is derived from *Archaeopterygium scintillans* (…). Archaeoglobus fulgidus CarB and CarRP originate from Mucor ( ); Mucor circinelloides ), CrtW originates from Chlamydomonas reinhardtii ( Chlamydomonas reinhardtii All of the above genes were synthesized artificially after codon optimization and appropriate avoidance of commonly used restriction enzyme sites.

[0051] Promoters, terminators, endogenous genes, and related upstream and downstream homologous sequences in *Saccharomyces cerevisiae*, including the CYC1 terminator, GAL10 promoter, GAL1 promoter, PGK1 terminator, ACT1 terminator, GPM1 terminator, TDH2 terminator, SpO1 terminator, ERG10 gene and ERG10 terminator, and the truncated 3-hydroxy-3-methylglutaryl-CoA reductase gene (tHMGR1), were obtained by PCR amplification using the genome of *Saccharomyces cerevisiae* strain BY4742 as a template, with suitable primers designed and synthesized. The upstream homologous sequence of LEU2 and the LEU2 marker were also obtained by PCR amplification from plasmid pRS405. The DR-KlURA3-DR nutrient tag sequence was obtained by PCR amplification using plasmid pWJ1042 (full genome sequence shown in SEQ ID NO:6) as a template.

[0052] (2) Construction of gene fragments

[0053] a) Construction of gene fragment 1

[0054] A 631bp homologous sequence upstream of the yeast TRP1 site, the CYC1 terminator, the CarB gene, the GAL10 promoter, the GAL1 promoter, the CarRP gene, the PGK1 terminator, and a 733bp homologous sequence downstream of the yeast TRP1 site were amplified and sequentially spliced ​​together using overlap extension PCR to obtain a fragment containing PmeI restriction sites at both ends: TRP1 LHA-TCYC1-CarB-PGAL10-PGAL1-CarRP-TPGK1-TRP1 RHA. This fragment was then ligated into the vector pJET1.2 (plasmid map shown). Figure 6 ), and the integrated plasmid of gene fragment 1 was obtained, denoted as:

[0055] pJET-TRP1-TCYC1-CarB-PGAL10-PGAL1-CarRP-TPGK1.

[0056] The integrative plasmid was transformed into competent E. coli DH5α cells, colony PCR was used for screening, and the plasmid was extracted for enzyme digestion and sequencing verification to ensure that the target fragment was correctly ligated and that the base sequence had not been mutated.

[0057] After verification, the gene fragment was cut with PmeI restriction endonuclease to obtain gene fragment 1, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0058] b) Construction of gene fragment 2

[0059] The following homologous sequences were amplified: a 394 bp upstream of the yeast ypl062w site, the DR-URA3-DR nutrient tag sequence, the CYC1 terminator, the CarB gene, the GAL10 promoter, the GAL1 promoter, the CarRP gene, the PGK1 terminator, and a 317 bp downstream of the yeast ypl062w site. These sequences were then sequentially spliced ​​together using overlap extension PCR to obtain the fragment ypl062w LHA-DR-URA3-DR-TCYC1-CarB-PGAL10-PGAL1-CarRP-TPGK1-ypl062w RHA, which contains PmeI restriction sites at both ends. This fragment was then ligated into the vector pJET1.2 to obtain the gene fragment 2 integrated plasmid, denoted as: pJET-ypl062w-DR-URA3-DR-TCYC1-CarB-PGAL10-PGAL1-CarRP-TPGK1.

[0060] The integrative plasmid was transformed into competent E. coli DH5α cells, colony PCR was used for screening, and the plasmid was extracted for enzyme digestion and sequencing verification to ensure that the target fragment was correctly ligated and that the base sequence had not been mutated.

[0061] After verification, the gene fragment was cut with PmeI restriction endonuclease to obtain gene fragment 2, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0062] c) Construction of gene fragment 3

[0063] A 426bp homologous sequence upstream of the yeast gal7 site, the DR-URA3-DR nutrient tag sequence, the ERG10 gene and its terminator, the GAL7 promoter, the ACT1 terminator, the tHMGR1 gene, the GAL10 promoter, the GAL1 promoter, the CrtE gene, the GPM1 terminator, and a 234bp homologous sequence downstream of the yeast gal1 site were amplified and sequentially spliced ​​together using overlap extension PCR to obtain a fragment containing PmeI restriction sites at both ends.

[0064] gal7LHA-DR-URA3-DR-TERG10-ERG10-PGAL7-TACT1-tHMGR1-PGAL10-PGAL1-CrtE-TGPM1-gal1 RHA. Then, it was ligated into the vector pJET1.2 to obtain the gene fragment 3 integration plasmid, denoted as:

[0065] pJET-gal-DR-URA3-DR-TERG10-ERG10-PGAL7-TACT1-tHMGR1-PGAL10-PGAL1-CrtE-TGPM1.

[0066] The integrative plasmid was transformed into competent E. coli DH5α cells, colony PCR was used for screening, and the plasmid was extracted for enzyme digestion and sequencing verification to ensure that the target fragment was correctly ligated and that the base sequence had not been mutated.

[0067] After verification, the gene fragment was cut with PmeI restriction endonuclease to obtain gene fragment 3, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0068] d) Construction of gene fragment 4

[0069] A 561bp homologous sequence upstream of the yeast LEU2 site, the LEU2 marker, the TDH2 terminator, the GAL1 promoter, the CrtW gene, the SpO1 terminator, and a 584bp homologous sequence downstream of the yeast LEU2 site were amplified and sequentially spliced ​​together using overlap extension PCR to obtain a fragment containing PmeI restriction sites at both ends: LEU2 LHA-LEU2-PGAL1-CrtW-TSPO1-LEU2 RHA. This fragment was then ligated into the vector pJET1.2 to obtain the gene fragment 4 integrated plasmid, denoted as pJET-LEU2-PGAL1-CrtW-TSPO1.

[0070] The integrative plasmid was transformed into competent E. coli DH5α cells, colony PCR was used for screening, and the plasmid was extracted for enzyme digestion and sequencing verification to ensure that the target fragment was correctly ligated and that the base sequence had not been mutated.

[0071] After verification, the gene fragment was cut with PmeI restriction endonuclease to obtain gene fragment 4, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0072] e) Construction of gene fragment 5

[0073] A 362bp homologous sequence upstream of the yeast gal80 site, the DR-URA3-DR nutrient tag sequence, the GAL1 promoter, the CrtW gene, the SpO1 terminator, and a 454bp homologous sequence downstream of the yeast gal80 site were amplified and sequentially spliced ​​together using overlap extension PCR to obtain a fragment containing PmeI restriction sites at both ends: gal80 LHA-DR-URA3-DR-PGAL1-CrtW-TSPO1-gal80 RHA. This fragment was then ligated into the vector pJET1.2 to obtain a gene fragment 5 integrated plasmid, denoted as: pJET-gal80-DR-URA3-DR-PGAL1-CrtW-TSPO1.

[0074] The integrative plasmid was transformed into competent E. coli DH5α cells, colony PCR was used for screening, and the plasmid was extracted for enzyme digestion and sequencing verification to ensure that the target fragment was correctly ligated and that the base sequence had not been mutated.

[0075] After verification, the gene fragment was cut with PmeI restriction endonuclease to obtain gene fragment 5, the nucleotide sequence of which is shown in SEQ ID NO.5.

[0076] The schematic diagrams of gene fragments 1-5 are shown below. Figures 1-5 .

[0077] (3) Construction of Saccharomyces cerevisiae MBP_Sc22

[0078] Gene fragment 1 was transformed into *Saccharomyces cerevisiae* CEN.PK2-1D using the lithium acetate method. The fragment integrated into the yeast genome through homologous recombination between the upstream and downstream homologous sequences of TRP1 and the trp1 site. After transformation, the transformants were screened using SD-TRP solid plates (6.7 g / L yeast nitrogen source, 20 g / L glucose, 2 g / L mixed amino acid powder lacking tryptophan, and 2% agar powder). The transformed strains were streaked and purified, and the yeast genome was extracted for PCR verification. The correctly verified recombinant strains were preserved as glycerol culture and named FNFH_Sc11.

[0079] Gene fragment 2 was transformed into *Saccharomyces cerevisiae* FNFH_Sc11 using the lithium acetate method. The fragment integrated into the genome through homologous recombination between the upstream and downstream homologous sequences of ypl062w and the ypl062w site on the yeast genome. After transformation, the transformants were screened on SD-TRP-URA solid plates (6.7 g / L yeast nitrogen source, 20 g / L glucose, 2 g / L mixed amino acid powder lacking tryptophan and uracil, 2% agar powder). Transformants were purified, and yeast genomes were extracted for PCR verification. Correctly verified recombinant strains were cultured on YPD liquid medium (20 g / L peptone, 20 g / L peptone, 10 g / L yeast extract). A small amount of the culture was then spread onto 5-fluoroorotic acid (5-FOA) solid plates (because the DR-URA3-DR nutrient tag has 1...). The yeast itself utilizes two identical 43bp repetitive sequences (DRs) to undergo homologous recombination, deleting the URA3 gene and one of the DRs. Strains containing URA3 can convert 5-FOA into a cytotoxic substance, preventing growth on media containing 5-FOA (thus screening for strains with deleted URA3). Single colonies are isolated, cultured, and their genomes are extracted for PCR verification to screen for the correct strains that have deleted the URA3 gene through spontaneous recombination between DR sequences. The verified recombinant strains are preserved as glycerol culture and named FNFH_Sc12.

[0080] Gene fragment 3 was transformed into *Saccharomyces cerevisiae* FNFH_Sc12 using the lithium acetate method. It integrated into the yeast genome through homologous recombination with the upstream and downstream homologous sequences of gal7 and gal1, respectively. After transformation, the transformants were screened using SD-TRP-URA agar plates (6.7 g / L yeast nitrogen source, 20 g / L glucose, 2 g / L mixed amino acid powder lacking tryptophan and uracil, and 2% agar powder). Transformants were purified, cultured, and their genomes were extracted for PCR verification. Correctly verified recombinant strains were cultured in YPD liquid medium, and a small amount of the culture was spread onto 5-FOA agar plates. Single colonies were picked, purified, and their genomes were extracted for PCR verification. Strains that correctly deleted the URA3 gene through spontaneous recombination between DR sequences were screened. The correctly verified recombinant strains were preserved as glycerol culture and named FNFH_Sc13.

[0081] Gene fragment 4 was transformed into *Saccharomyces cerevisiae* FNFH_Sc13 using the lithium acetate method. The fragment integrated into the genome through homologous recombination between the upstream and downstream homologous sequences of LEU2 and the leu2 site on the yeast genome. After transformation, the transformants were screened using SD-TRP-LEU solid plates (6.7 g / L yeast nitrogen source, 20 g / L glucose, 2 g / L mixed amino acid powder lacking tryptophan and leucine, and 2% agar powder). The transformed strains were streaked and purified, and the yeast genome was extracted for PCR verification. The correctly verified recombinant strains were preserved as glycerol culture and named FNFH_Sc17.

[0082] Gene fragment 5 was transformed into *Saccharomyces cerevisiae* FNFH_Sc17 using the lithium acetate method. It integrated into the yeast genome through homologous recombination between the upstream and downstream homologous sequences of gal80 and the gal80 site. After transformation, the transformants were screened using SD-TRP-LEU-URA agar plates (6.7 g / L yeast nitrogen source, 20 g / L glucose, 2 g / L mixed amino acid powder lacking tryptophan, leucine, and uracil, 2% agar powder). Transformants were purified, and yeast genomes were extracted for PCR verification. Correctly verified recombinant strains were cultured in YPD liquid medium, and a small amount of the culture was spread onto 5-FOA agar plates. Single colonies were picked, purified, and their genomes were extracted for PCR verification. Strains that correctly deleted the URA3 gene through spontaneous recombination between DR sequences were screened. The correctly verified recombinant strains were preserved as glycerol culture and named MBP_Sc22.

[0083] This strain was deposited on September 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The accession number is CGMCC No. 35982, and the suggested name is *Saccharomyces cerevisiae*. Saccharomyces cerevisiae ).

[0084] 2. Fermentation of MBP_Sc22

[0085] 10 μL of MBP_Sc22 glycerol bacteria was inoculated into 5 mL of YPD liquid medium and activated overnight at 30 ℃ and 250 rpm. 2% (v / v) of activated seed culture was then inoculated into 50 mL of YPD liquid medium and incubated at 30 ℃ and 250 rpm for 36 h to obtain the MBP_Sc22 fermentation broth.

[0086] 3. Extraction and Detection of Fermentation Products

[0087] Two equal volumes of MBP_Sc22 fermentation broth were collected, centrifuged at 12000 rpm for 2 min, and the cells were washed twice with water. One portion of the cells was dried at 80 ℃ to constant weight, and the dry weight of the cells was obtained. The other portion of the cells was resuspended in 1 mL of 3M HCl, boiled in a boiling water bath for 2 min, and then placed on ice for 3 min. The lysed cells were centrifuged at 12000 rpm at 4 ℃ for 2 min, the supernatant was discarded, and the cells were washed twice with water. 1 mL of acetone was added, vortexed for 5 min, and then centrifuged to collect the acetone phase. The acetone phase was filtered through a 2 µm organic filter membrane for high-performance liquid chromatography (HPLC) analysis (Agulent 1260 Infinity, UV detector). The analysis was performed using a Hypersil GOLD™ C18 column (150 × 4.6 mm, 5 μm) with methanol-acetonitrile-dichloromethane (21:21:8, v / v) as the mobile phase, a flow rate of 1.0 mL / min, a column temperature of 30 ℃, and cantharidin was detected at a wavelength of 470 nm.

[0088] III. Experimental Results

[0089] like Figure 8 As shown, after 36 h of fermentation, the MBP_Sc22 culture solution turned orange-red and accumulated a large amount of canthaxanthin, with a canthaxanthin yield of 14.9 mg / g DCW.

[0090] Example 2: Preparation and characterization of a norepinephrine-encapsulated cantharidin-producing engineered yeast preparation (NE-CX-Sc)

[0091] I. Experimental Materials

[0092] (1) Microbial strains

[0093] The engineered yeast MBP_Sc22, which produces cantharidin, was designated CX-Sc in subsequent experiments.

[0094] (2) Culture medium

[0095] YPD liquid culture medium: glucose 25 g / L, peptone 20 g / L, yeast extract 10 g / L.

[0096] YPD solid plates: glucose 25 g / L, peptone 20 g / L, yeast extract 10 g / L, agar 20 g / L.

[0097] II. Experimental Methods

[0098] (1) Activation of CX-Sc

[0099] 10 μL of CX-Sc glycerol bacteria was inoculated into 5 mL of YPD liquid medium and cultured overnight at 30 ℃ and 250 rpm. Then, 2% (v / v) of activated seed culture was inoculated into 50 mL of YPD liquid medium and cultured at 30 ℃ and 250 rpm for 36 h. Before use, the culture was centrifuged twice with 0.9% physiological saline (4 ℃, 5000 rpm, 3 min), and resuspended in 0.9% physiological saline to a final concentration of 4 × 10⁻⁶. 8 CFU / mL, for later use.

[0100] (2) Preparation of a cantharidin-encapsulated probiotic preparation containing norepinephrine.

[0101] Add norepinephrine (NE) to sterile PBS to prepare norepinephrine solutions with final concentrations of 0.5 mg / mL and 1 mg / mL (ultrasound-assisted dissolution). Add CX-Sc (4×10⁻⁶) to sterile PBS. 8 CFU was resuspended in 4 mL of 0.5 mg / mL and 1 mg / mL NE solution, respectively, and incubated at 37 °C and 100 rpm for 3 h. The solutions were washed twice with 0.9% physiological saline and then resuspended in 0.9% physiological saline to a final concentration of 4 × 10⁻⁶. 8 CFU / mL yielded NE-encapsulated cantharidin-producing engineered yeasts NE-CX-Sc and NE-CX-Sc2.

[0102] (3) Quantitative analysis of NE on the surface of engineered probiotic preparations

[0103] Neoplasm (NE) on the surface of NE-CX-Sc was quantified using a protein quantification assay (BCA) kit. In short, 20 μL of diluted samples (CX-Sc, NE-CX-Sc, and NE-CX-Sc2) were added to 200 μL of reaction solution, incubated at 37 °C for 30 min, and the absorbance was measured at 562 nm. A standard curve was plotted using NE as a standard. CX-Sc was used as a blank control to quantify the NE coating on the surfaces of NE-CX-Sc and NE-CX-Sc2.

[0104] (4) Effect of NE coating on the activity of engineered yeast

[0105] The samples (CX-Sc, NE-CX-Sc and NE-CX-Sc2) were serially diluted with 0.9% physiological saline, and 100 μL of each diluted bacterial solution was spread on YPD solid plates. The plates were incubated upside down in a 30 ℃ incubator for 2 days, and plate counts were performed to analyze the effect of NE coating on the activity of engineered yeast.

[0106] (5) Measurement of zeta potential on the surface of engineered probiotic preparations

[0107] The samples (CX-Sc and NE-CX-Sc) were diluted and dispersed in deionized water, and the zeta potentials on the CX-Sc and NE-CX-Sc surfaces were determined by measuring electrophoretic light scattering using a nanoparticle size potentiometer (ZetaSizer Nano-Sizer 90).

[0108] (6) Characterization by scanning electron microscopy (SEM)

[0109] Samples (CX-Sc, NE-CX-Sc, and NE-CX-Sc2) were added to 2.5% glutaraldehyde and fixed at 4 °C for 24 h. The fixative was removed by centrifugation, and the samples were washed three times with PBS. The samples were then dehydrated using a gradient of ethanol (50%, 70%, 80%, 90%) (15 min / time), followed by three dehydration cycles of 100% ethanol (30 min / time). After treatment with 100% tert-butanol three times (30 min / time), and freeze-dried, the samples were fixed to the sample stage using conductive carbon adhesive and sputtered with gold for approximately 30 s. The images were then observed and analyzed under a scanning electron microscope (Regulus-8100).

[0110] (7) Biological transmission electron microscopy (TEM) characterization

[0111] Samples (CX-Sc, NE-CX-Sc, and NE-CX-Sc2) were fixed in 2.5% glutaraldehyde at 4 °C for 24 h. After centrifugation to remove the fixative, the samples were washed three times with PBS and then fixed with 1% osmium tetroxide solution for 1 h. After centrifugation to remove the fixative, the samples were washed three times with PBS. The samples were then dehydrated using a gradient of ethanol (30%, 50%, 70%, 90%, 100%) (20 min / time), and finally dehydrated twice with 100% acetone (20 min / time). After embedding and permeation treatment, ultrathin sections of 70-90 nm were prepared and placed on a copper grid. The sections were then stained sequentially with uranyl acetate and lead citrate, dried, and observed under a transmission electron microscope (HITACHI HT7800) for image analysis.

[0112] III. Experimental Results

[0113] (1) Preparation of NE-CX-Sc

[0114] To improve the efficiency of oral delivery of engineered yeast probiotic drugs to the colon, norepinephrine was used to encapsulate engineered yeast CX-Sc. For example... Figure 9 As shown in Figures A and B, the self-polymerization of norepinephrine (NE) is an oxidative darkening process. With increasing NE concentration, the bacterial solution darkens, and the amount of NE polymerized on the surface of the engineered bacteria also increases. This indicates that the efficiency of norepinephrine encapsulation of engineered bacteria is positively correlated with concentration. However, prolonged exposure of engineered yeast to the encapsulation solution environment and over-encapsulation may lead to a decrease in their viability. Figure 9 As shown in Figure C, the activity of engineered yeast was negatively correlated with the concentration of NE. Specifically, coating engineered yeast with NE at a concentration of 0.5 mg / mL for 3 h had no significant effect on its activity. Therefore, the concentration of NE working solution for coating engineered yeast was determined to be 0.5 mg / mL, and the coating time was determined to be 3 h. Under these conditions, the surface Zeta potential of NE-CX-Sc engineered yeast increased from -27.2 mV to -23.2 mV after NE coating. Figure 9 (D in the text). These results indicate that 0.5 mg / mL of NE can encapsulate engineered yeast cells while maintaining their viability.

[0115] (2) Ultrastructural characterization of NE-CX-Sc

[0116] To further understand the surface morphology of NE-CX-Sc, SEM and TEM were used to characterize NE-CX-Sc. Figure 10 As shown, the CX-Sc surface is smooth, while a continuous, dense black oxide layer appears on the CX-Sc cell surface after NE coating, and the roughness of the oxide layer increases with increasing NE concentration. Notably, a large number of polynorepinephrine particles appeared on the surface of the engineered yeast coated with NE at a concentration of 1 mg / mL, which may be the main reason for the decreased activity of the engineered yeast. These results indicate that NE has been successfully coated on the CX-Sc surface, and its surface microstructure exhibits a concentration-dependent change.

[0117] Example 3: Evaluation of Gastrointestinal Environment Tolerance of NE-CX-Sc

[0118] I. Experimental Materials

[0119] (1) Microbial strains

[0120] Cantharidin-producing engineered yeast MBP_Sc22 (CX-Sc) and norepinephrine-encapsulated cantharidin-producing engineered yeast (NC-CX-Sc).

[0121] (2) Simulated digestive fluid

[0122] Artificial gastric juice (SGF): sodium chloride 2 g / L, pepsin 3.2 g / L, pH adjusted to 1.5, filtered through a 0.22 μm aqueous sterile filter membrane.

[0123] Artificial intestinal fluid (SIF): potassium dihydrogen phosphate 6.8 g / L, trypsin 10 g / L, porcine bile salts 4 g / L, pH adjusted to 6.8 with 0.1 mol / L sodium hydroxide, filtered through a 0.22 μm aqueous sterile filter membrane.

[0124] II. Experimental Methods

[0125] Equal amounts (4×10) 8 CX-Sc and NE-CX-Sc (CFU) were added to SGF and SIF, respectively, and incubated at 37 ℃ and 100 rpm. At predetermined time points (0, 0.5, 1, 1.5, and 2 h), excess digestive fluid was removed from the samples, and 100 μL of the diluted bacterial solution was spread onto YPD solid plates and incubated upside down in a 30 ℃ incubator for 2 days. Plate counts were performed to evaluate the protective effect of the NE protective layer on engineered yeast under in vitro simulated gastrointestinal digestive fluid damage conditions.

[0126] III. Experimental Results

[0127] (1) Evaluation of the in vitro resistance of NE-CX-Sc to digestive fluid

[0128] Harsh physiological factors in the gastrointestinal tract, such as gastric acid, digestive enzymes, and bile salts, can significantly reduce the intestinal survival rate and shorten the intestinal retention time of orally administered engineered probiotics, ultimately affecting treatment efficacy. To verify whether NE encapsulation can improve the survival rate of engineered yeast in the gastrointestinal tract, the survival of CX-Sc and NE-CX-Sc in in vitro simulated digestive environments (SGF and SIF) was compared. Figure 11 As shown, compared to SIF (neutral pH + trypsin + 0.4% bile salts), SGF (low pH + pepsin) had a greater impact on the activity of engineered yeast (CX-Sc and NE-CX-Sc), and the survival rate of NE-CX-Sc was significantly higher than that of CX-Sc in both SGF and SIF. These results indicate that engineered yeast is highly sensitive to SGF, while NE encapsulation can significantly improve the survival ability of engineered yeast in the gastrointestinal environment.

[0129] Example 4: Evaluation of NE-CX-Sc's targeting ability on macrophages

[0130] I. Experimental Materials

[0131] (1) Microbial strains

[0132] Cantharidin-producing engineered yeast MBP_Sc22 (CX-Sc) and norepinephrine-encapsulated cantharidin-producing engineered yeast (NC-CX-Sc).

[0133] (2) Cells

[0134] Macrophages: RAW264.7.

[0135] (3) Cell culture medium

[0136] RAW264.7 cell culture medium: 89% DMEM high glucose medium + 10% fetal bovine serum + 1% penicillin-streptomycin mixture (v / v).

[0137] II. Experimental Methods

[0138] After resuscitation, RAW264.7 cells were placed in RAW264.7 cell culture medium and cultured at 37 ℃ in a 5% CO2 incubator. Cells were passaged after reaching 80% confluence. Red fluorescent protein (RFP)-labeled CX-SC and NE-CX-Sc were added to RAW264.7 cells treated with 1 μg / mL lipopolysaccharide (LPS), and incubated at 37 ℃ for 3 h. Cells were washed three times with PBS, and the nuclei were stained with DAPI to remove excess dye. The cells were then observed under a fluorescence microscope to evaluate the targeting ability of CX-Sc and NE-CX-Sc to macrophages.

[0139] III. Experimental Results

[0140] The ability of β-1,3-D-glucan in the yeast cell wall to be recognized by Dectin-1 and to target macrophages is one of its key advantages as an oral engineered probiotic. After co-culturing LPS-treated macrophages with an RFP-labeled engineered bacterial preparation for 3 h, uptake of CX-Sc and NE-CX-Sc by macrophages was detected in both cases. Figure 12 The results showed that NE encapsulation did not disrupt the macrophage targeting ability of engineered yeast mediated by β-1,3-D-glucan and Dectin-1. NE-CX-Sc can achieve targeted localization of inflammatory sites by leveraging the macrophage recruitment characteristics of inflammatory sites.

[0141] Example 5: Evaluation of Gastrointestinal Adhesion of NE-CX-Sc

[0142] I. Experimental Materials

[0143] (1) Microbial strains

[0144] Cantharidin-producing engineered yeast MBP_Sc22 (CX-Sc) and norepinephrine-encapsulated cantharidin-producing engineered yeast (NC-CX-Sc).

[0145] (2) Cells

[0146] Human colorectal adenocarcinoma epithelial cells: Caco-2 cells.

[0147] (3) Cell culture medium

[0148] Caco-2 cell culture medium: 79% DMEM medium + 20% fetal bovine serum + 1% penicillin-streptomycin mixture (v / v).

[0149] (4) Laboratory animals

[0150] Male C57BL / 6J mice (non-specific pathogens, 6-8 weeks old, 20.0±2.0 g) were housed in a controlled environment (temperature 22±2 ℃, humidity 55±5%) with a 12 h light / dark cycle and free access to food and water.

[0151] II. Experimental Methods

[0152] (1) Evaluation of the adhesion ability of NE-CX-Sc to colonic epithelial cells

[0153] After resuscitation, Caco-2 cells were placed in Caco-2 cell culture medium and cultured at 37 ℃ in a 5% CO2 incubator. Cells were passaged after reaching 80% confluence. RFP-labeled CX-SC and NE-CX-Sc were added to Caco-2 cells, incubated at 37 ℃ for 3 h, washed three times with PBS, and the nuclei were stained with DAPI. After washing away excess dye, the cells were observed under a fluorescence microscope to evaluate the adhesion ability of CX-Sc and NE-CX-Sc to colonic epithelial cells.

[0154] (2) Retention of NE-CX-Sc in the gastrointestinal tract of mice

[0155] CX-SC and NE-CX-Sc (4×10) marked with RFP 8 C57BL / 6J mice were administered CFU / mouse via gavage. Four hours later, the gastrointestinal tract of the mice was collected and imaged using a small animal in vivo optical three-dimensional imaging system (IVIS, Lumina III). The fluorescence signal in the target area was quantified using IVISLiving Image 4.2 software.

[0156] III. Experimental Results

[0157] (1) Adhesion of NE-CX-Sc to Caco-2

[0158] Caco-2 cells share characteristics with mature intestinal cells and have been widely used in probiotic adhesion studies. After co-culturing Caco-2 cells with an RFP-labeled engineered bacterial preparation for 3 hours, the fluorescence images are as follows... Figure 13As shown, CX-Sc showed almost no adhesion to Caco-2 cells, a result consistent with the study by Sun et al. (Sun et al., 2020) where yeast dextran particles (YGP) were almost unable to be internalized into colonic epithelial cells. In contrast, NE-CX-Sc exhibited significant adhesion to Caco-2 cells, suggesting that NE encapsulation can significantly enhance the adhesion of engineered yeast to colonic epithelial cells, thereby helping to prolong its retention time in the intestine.

[0159] (2) Retention of NE-CX-Sc in the gastrointestinal tract of mice

[0160] Based on the strong digestive fluid resistance and adhesion of NE-coated engineered yeast (NE-CX-Sc) in in vitro experiments, further investigation was conducted on its retention in the mouse gastrointestinal tract. Four hours after oral administration of RFP-labeled engineered bacterial preparations (CX-Sc and NC-CX-Sc) to mice, intestinal IVIS images were obtained. Figure 14 As shown in Figures A and B), the fluorescence intensity of the NE-CX-Sc group was significantly higher than that of the CX-Sc group. This indicates that NE encapsulation can effectively enhance the retention and survival rate of engineered yeast in the mouse gastrointestinal tract, thus providing an important guarantee for its continued biological function in the intestine.

[0161] Example 6: Evaluation of the therapeutic effect of NE-CX-Sc on DSS-induced colitis in mice

[0162] I. Experimental Materials

[0163] (1) Microbial strains

[0164] Cantharidin-producing engineered yeast MBP_Sc22 (CX-Sc), norepinephrine-encapsulated cantharidin-producing engineered yeast (NC-CX-Sc), and common yeast (Sc).

[0165] (2) Laboratory animals

[0166] Male C57BL / 6J mice (non-specific pathogens, 6-8 weeks old, 20.0±2.0 g) were housed in a controlled environment (temperature 22±2 ℃, humidity 55±5%) with a 12 h light / dark cycle and free access to food and water.

[0167] II. Experimental Methods

[0168] (1) Animal experiment procedure

[0169] After one week of acclimatization, 30 male C57BL / 6J mice were randomly divided into 5 groups (n=6 per group): blank control group (NC), model control group (MC), norepinephrine-encapsulated cantharidin-engineered yeast group (NE-CX-Sc), cantharidin-engineered yeast group (CX-Sc), and ordinary yeast group (Sc). Mice in the NC group were provided with regular drinking water throughout the experiment, while the other groups were provided with free access to 2.5% dextran sulfate sodium (DSS) for 6 days. From the fourth day of modeling, the corresponding microbial preparation groups (NE-CX-Sc, CX-Sc, and Sc groups) were administered the corresponding microbial preparation (4×10⁻⁶). 8 The mice were administered an equal volume of saline via gavage until day 10. Feces were collected, and the mice were fasted. On day 11, all mice were euthanized, and experimental samples were collected for subsequent experiments.

[0170] (2) Disease Activity Index (DAI) in mice and its related scoring scheme

[0171] During the experiment, changes in body weight, fecal viscosity, and fecal blood in each group of mice were recorded daily. Based on the standardized scoring system shown in Table 1, the disease activity index of each group of mice was systematically evaluated.

[0172] Table 1. Disease Activity Index Scores for Mice

[0173] score weight loss fecal viscosity blood in stool 0 ＜1% normal normal 1 1-5% - - 2 5-10% Unformed stool Small amount of rectal bleeding 3 10-15% - - 4 ＞15% diarrhea Massive rectal bleeding .

[0174] (3) Colonic H&E staining

[0175] The prepared colon paraffin sections were stained with hematoxylin (3-5 min), rinsed to remove excess stain, differentiated with differentiation solution, washed with water, blued with blue solution, rinsed with running water, dehydrated with 95% alcohol (1 min), stained with eosin (15 s), and mounted with neutral resin after dehydration.

[0176] (4) Reactive oxygen species (ROS) staining

[0177] After embedding frozen colon tissue, frozen sections (10 μm) were prepared, thawed, and air-dried. The sections were fixed with methanol for 10 min, washed three times with PBS, and then spun dry. Diethidium hydrogen sulfide (DHE) staining solution (1:1000) was added, and the sections were incubated at 37 ℃ for 30 min. Excess staining solution was washed away, cell nuclei were counterstained with DAPI, and the sections were mounted with anti-fluorescence quenching mounting medium.

[0178] (5) Biochemical index determination

[0179] The colon, frozen at -80 °C, was thawed on ice. 0.1 g of colon tissue was weighed and added to 0.9 mL of physiological saline. A colon tissue homogenate was prepared under ice bath conditions and centrifuged (4 °C, 10000 rpm, 10 min) to collect the supernatant. The protein concentration of the colon homogenate was determined according to the method described in the BCA kit instructions. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-10 (IL-10), and interleukin-6 (IL-6) in the colon tissue homogenate were determined according to the method provided by the manufacturer of the enzyme-linked immunosorbent assay (ELISA) kit.

[0180] III. Experimental Results

[0181] To further investigate the therapeutic effect of NE-CX-Sc on colitis, a mouse colitis model was established by free drinking of 2.5% DSS, and each treatment group was given corresponding microbial preparations for intervention. Figure 15 The fecal characteristics, occult blood status, and weight changes of mice in each group were recorded, and the DAI index was evaluated in conjunction with Table 1.

[0182] like Figure 15 As shown in Figure C, the body weight of mice in the NC group showed a slow upward trend, while the body weight of mice in the other groups showed a downward trend starting from day 5 after DSS intervention. By day 10 of the experiment, compared with the MC group, the body weight loss of mice in the NE-CX-Sc group and CX-Sc group was significantly reduced, while the body weight loss of mice in the Sc group was not significantly different from that in the MC group. Notably, NE-CX-Sc was significantly more effective than CX-Sc and Sc groups in improving body weight loss in UC mice. In addition, compared with the NC group, mice in the MC group had loose stools, obvious bloodstains around the anus, strong positive fecal occult blood, and significantly increased DAI scores, while the intervention of NE-CX-Sc significantly reversed these adverse changes. Figure 15 (B and D in the above). The results show that the MC group mice exhibited typical pathological features of colitis, including loose stools, bloody stools, continuous weight loss and significantly increased DAI scores, indicating that the mouse colitis model was successfully established. NE-CX-Sc significantly reversed the above pathological manifestations, and its intervention effect was significantly better than other experimental groups.

[0183] A shortened colon is a direct reflection of intestinal tissue damage and repair. For example... Figure 15As shown in E and F, compared to the NC group (6.12±0.75 cm), the colon length of mice in the MC group was significantly shortened (4.05±0.19 cm). The intervention of microbial agents improved this pathological symptom to varying degrees. The colon lengths of mice in the NE-CX-Sc, CX-Sc, and Sc groups were 5.60±0.42 cm, 4.82±0.20 cm, and 4.22±0.12 cm, respectively. The NE-CX-Sc group showed the best improvement, with its colon length returning to a level not significantly different from the NC group. Furthermore, histopathological analysis of the colon tissue showed (…). Figure 15 In the MC group mice treated with G and DSS, the colonic mucosa was significantly damaged, specifically manifested as: destruction or even disappearance of crypt structures, a large number of goblet cells missing, inflammatory cell infiltration, and thickening of the muscular layer. In contrast, NE-CX-Sc, CX-Sc, and Sc interventions alleviated colonic tissue damage to varying degrees. Among them, the NE-CX-Sc group showed the best effect in improving colonic tissue damage, characterized by intact colonic mucosa and crypt structures, sufficient number of goblet cells, and almost no inflammatory cell infiltration. These results indicate that NE-CX-Sc can effectively reverse colonic damage in colitis mice.

[0184] Excessive ROS can trigger oxidative stress and amplify inflammatory responses, leading to intestinal mucosal damage and pathogen invasion, which in turn overactivates the immune response, ultimately promoting the development of intestinal inflammation. ROS staining results showed that, compared with the MC group, the NE-CX-Sc group had significantly lower ROS fluorescence intensity in the colonic tissue of mice, indicating that NE-CX-Sc can effectively reduce ROS levels in the colonic tissue of UC mice. Figure 16 Furthermore, to further elucidate the regulatory role of NE-CX-Sc in intestinal inflammation in UC mice, the expression levels of key inflammatory factors in colon tissue were simultaneously detected, and the results are as follows: Figure 17 As shown, after DSS intervention, the expression levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in the colonic tissue of MC group mice were significantly increased, while the expression level of anti-inflammatory factor IL-10 was significantly decreased. NE-CX-Sc intervention effectively reversed this inflammatory imbalance, and its therapeutic effect was significantly better than that of the CX-Sc group and the Sc group. This suggests that NE-CX-Sc can not only alleviate oxidative stress by downregulating ROS, but also significantly inhibit the excessive production of pro-inflammatory factors and significantly promote the expression of anti-inflammatory factor IL-10, thereby effectively regulating the balance of the intestinal inflammatory microenvironment.

[0185] Example 7: Biosafety Evaluation of NE-CX-Sc

[0186] I. Experimental Materials

[0187] (1) Microbial strains

[0188] Norepinephrine-encapsulated cantharidin-producing engineered yeast (NC-CX-Sc).

[0189] (2) Laboratory animals

[0190] Male C57BL / 6J mice (non-specific pathogens, 6-8 weeks old, 20.0±2.0 g) were housed in a controlled environment (temperature 22±2 ℃, humidity 55±5%) with a 12 h light / dark cycle and free access to food and water.

[0191] II. Experimental Methods

[0192] Male C57BL / 6J mice were tested after oral administration of NE-CX-Sc (4×10⁻⁶) for 6 consecutive days. 8 After CFU / mouse / day, the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE), and blood urea nitrogen (BUN) in mice were measured.

[0193] III. Experimental Results

[0194] The biosafety of engineered probiotics is a core prerequisite for their practical application in clinical settings, food, and animal feed. After continuous oral administration of NE-CX-Sc for 6 days, key indicators of liver and kidney function in mice were as follows... Figure 18 As shown, the serum levels of ALT, AST, CRE, and BUN in mice treated with NE-CX-Sc were not significantly different from those in normal mice, indicating that the constructed NE-CX-Sc did not cause adverse reactions in liver and kidney function and had good biosafety.

[0195] Example 8: Evaluation of the regulatory effect of NE-CX-Sc on the intestinal flora of colitis mice

[0196] I. Experimental Materials

[0197] Mouse feces.

[0198] II. Experimental Methods

[0199] Genomic DNA was extracted from fecal samples using a commercial DNA extraction kit. The V3-V4 hypervariable region of the 16S rDNA was amplified by PCR using universal primer pairs (338F: 5'-ACTCCTACGGGAGGCAGCAG-3′ (SEQ ID NO.7)); 806R: 5'-GGACTACHVGGTWTCTAAT-3′ (SEQ ID NO.8)). The sequence was optimized by Meiji Biotechnology Co., Ltd. (Shanghai, China) to obtain the final reads.

[0200] III. Experimental Results

[0201] The gut microbiome, as an important component of the intestinal barrier, plays a crucial role in the occurrence and development of intestinal inflammation. To clarify the therapeutic mechanism of NE-CX-Sc, 16S rRNA sequencing was used to further evaluate its impact on the gut microbiota of UC mice. α-diversity analysis showed that NE-CX-Sc intervention significantly reversed the DSS-induced decline in gut microbiota abundance and diversity. Figure 19 (A and B in the text). Furthermore, genus-level analysis showed that NE-CX-Sc significantly increased the levels of Bacteroides (A and B) in the gut of UC mice. Bacteroides ) and Akkermania ( Akkermansia The relative abundance of Shigella ( ) was reduced, while the abundance of Shigella ( ) was decreased. Escherichia-Shigella The relative abundance of ) Figure 19 (C, D, and E in the text). Among them, *Bacteroides* can improve intestinal function by participating in nutrient metabolism to produce short-chain fatty acids and competing with pathogenic bacteria for adhesion sites; *Ackermania* can promote the expression of tight junction proteins, induce the secretion of the anti-inflammatory factor IL-10, and promote the repair of damaged intestinal mucosa; *Shigella* is a genus of enteropathogenic bacteria that can increase the risk of bacterial dysentery in humans by altering intestinal epithelial physiology and tissue invasiveness. In summary, NE-CX-Sc can reverse the decline in intestinal flora abundance and diversity in DSS-induced colitis mice and restore the intestinal microbiota structure by enriching beneficial bacteria and inhibiting harmful bacteria, thereby achieving the goal of treating colitis.

Claims

1. A method for preparing an adhesive engineered yeast probiotic preparation, characterized in that, The specific steps include: adding norepinephrine to sterile PBS to prepare a solution, and then adding brewer's yeast ( Saccharomyces cerevisiae MBP_Sc22 was resuspended in norepinephrine solution, shaken and incubated at room temperature, washed and resuspended with physiological saline to obtain the final product. The preservation number of the brewer's yeast is CGMCC No. 35982; the concentration of norepinephrine is 0.5 mg / mL, and the encapsulation time is 3 h.

2. The method as described in claim 1, characterized in that, The brewing yeast ( Saccharomyces cerevisiae The concentration of MBP_Sc22 was 3.5 × 10⁻⁶. 8 -4.5×10 8 CFU / mL.

3. An adhesive engineered yeast probiotic preparation is prepared by the method according to any one of claims 1-2.

4. The application of the adhesive engineered yeast probiotic preparation as described in claim 3 in the preparation of products for treating colitis.

5. The use of the adhesive engineered yeast probiotic preparation as described in claim 3 in the preparation of products for regulating the intestinal flora of colitis.

6. A product for treating colitis, characterized in that, A probiotic preparation containing the adhesive engineered yeast of claim 3.

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