An engineered strain for intestinal delivery of glp-1 / gip dual receptor agonist and application thereof

Abstract

This invention relates to an engineered bacterial strain for intestinal delivery of a GLP-1 / GIP dual receptor agonist and its application. The strain uses *Saccharomyces boulardii*, a uracil-deficient trophoblastic yeast strain, as its chassis strain. The genome of this chassis strain integrates a polypeptide expression module. This module includes a secretion signal peptide coding sequence and a polypeptide coding sequence fused to the secretion signal peptide coding sequence. The polypeptide coding sequence encodes a polypeptide formed by multiple glucagon-like peptide-1 repeat units and multiple glucose-dependent insulinotropic peptide repeat units tandemly. Enzyme cleavage sites, which can be cleaved by yeast endogenous proteases, are positioned between adjacent repeat units, allowing the polypeptide to be cleaved in the Golgi apparatus or during secretory processing, releasing mature GLP-1 and GIP agonists. This strain enables orally, sustainably, and precisely dose-controlled delivery of the GLP-1 / GIP dual agonist, thereby achieving effective improvement in obesity and metabolic disorders, and enhancing drug adherence and safety.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to an engineered strain for intestinal delivery of a GLP-1 / GIP dual receptor agonist and its application. Background Technology

[0002] Obesity and its related metabolic diseases (including type 2 diabetes, fatty liver, and cardiovascular metabolic disorders) have become a major global public health problem, with their incidence continuing to rise and seriously threatening human health. In recent years, glucagon-like peptide-1 (GLP-1) receptor agonists have shown clear efficacy in weight control and blood glucose management; furthermore, dual agonists targeting both GLP-1 and glucose-dependent insulinotropic peptide (GIP) receptors can produce a synergistic effect, exhibiting stronger weight loss and metabolic improvement effects compared to single receptor agonists.

[0003] However, existing GLP-1 or GLP-1 / GIP-related treatments mostly use injectable administration or oral formulations with low bioavailability. Although injectable administration (subcutaneous or intravenous injection) has high bioavailability, it requires long-term regular injections, resulting in poor patient compliance. The developed oral formulations are mostly chemically modified small molecule or peptide formulations, which are prone to dose-limiting gastrointestinal adverse reactions (such as nausea, vomiting, diarrhea, anorexia, etc.) in clinical practice, affecting compliance. In addition, weight rebound and metabolic benefit decay after drug withdrawal are also relatively common.

[0004] While some GLP-1 / GIP analogues have been modified for longer-acting applications, such as the WO2025180387A1 approach, these still rely on injection and do not address the limitations of oral delivery. Therefore, there is an urgent need for a GLP-1 / GIP dual agonist delivery system that can provide a sustained, gradual, controllable, and better-tolerated delivery method to improve the accessibility and stability of long-term treatment. Summary of the Invention

[0005] This invention aims to at least partially address one of the technical problems in the related art. Therefore, the object of this invention is to provide an engineered strain for intestinal delivery of a GLP-1 / GIP dual receptor agonist and its application. This engineered strain enables orally, continuously, and precisely dose-controlled delivery of the GLP-1 / GIP dual agonist, thereby achieving effective improvement in obesity and metabolic disorders, and enhancing medication adherence and safety.

[0006] Therefore, in one aspect of the present invention, an engineered strain for intestinal delivery of GLP-1 / GIP dual receptor agonists is proposed. The engineered strain is based on a uracil-deficient *Saccharomyces boulardii* strain, and the genome of the base strain integrates a polypeptide expression module. The polypeptide expression module includes a secretion signal peptide coding sequence and a polypeptide coding sequence fused to the secretion signal peptide coding sequence. The polypeptide coding sequence encodes a polypeptide formed by multiple glucagon-like peptide-1 repeat units and multiple glucose-dependent insulinotropic peptide repeat units tandemly. Enzyme cleavage sites that can be cleaved by yeast endogenous proteases are provided between adjacent repeat units, so that the polypeptide can be cleaved in the Golgi apparatus or during secretory processing, releasing mature GLP-1 and GIP agonists.

[0007] The engineered yeast strain for intestinal delivery of GLP-1 / GIP dual receptor agonists according to the present invention uses *Saccharomyces boulardii* auxotrophic strain as the chassis strain, integrating an expression module containing the coding sequences for secretory signal peptides and polypeptides into its genome to obtain engineered probiotic yeast. The polypeptide coding sequence consists of multiple GLP-1 and GIP repeat units tandemly, with cleavage sites between adjacent repeat units that can be recognized by yeast endogenous proteases, allowing the polypeptide to be cleaved into mature active peptides and released extracellularly during secretory processing. Thus, continuous local secretion of GLP-1 / GIP agonists can be achieved locally in the intestine via oral administration of engineered probiotic yeast, resulting in gradual drug exposure, effectively reducing the risk of gastrointestinal adverse reactions caused by excessively high peak concentrations, while avoiding the pain and inconvenience of injection administration, thus improving patient compliance. Furthermore, the output ratio of the dual agonists can be programmably controlled by adjusting the copy number ratio of GLP-1 and GIP repeat units, avoiding ratio drift and batch-to-batch inconsistencies caused by colonization differences in multi-strain cocktail formulations, supporting personalized prescription design. Experiments have shown that this engineered strain can significantly reduce body weight and improve oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) indicators in a high-fat diet-induced obesity model, demonstrating a clear intervention effect on obesity and related metabolic disorders, and has broad prospects for clinical application.

[0008] In a second aspect, the present invention provides a method for preparing the above-mentioned engineered strain, comprising the following steps: Step 1: Construct a donor vector containing the polypeptide expression module; Step 2: The donor vector and the PBase transposase expression vector are jointly introduced into the chassis strain to obtain the recombinant strain; Step 3: Screen positive clones of the recombinant strain on uracil-deficient medium. After passage, the PBase transposase expression vector is removed from the positive clones to obtain stable integrated engineered strains.

[0009] According to the method of the present invention, using uracil-deficient *Saccharomyces boulardii* as the chassis strain, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) expression modules in different tandem ratios are integrated into the genome of the chassis strain using a transposon system to obtain a single-strain co-expressing recombinant strain of the dual agonist. This allows the engineered strain to continuously biosynthesize and secrete GLP-1 and GIP dual agonists in the intestine. The modular design of the donor vector allows for flexible replacement of the polypeptide coding sequence, adapting to the modification needs of different GLP-1 / GIP repeat unit ratios or variants, providing a convenient technical platform for subsequent optimization of the dual agonist ratio and expansion of the product pipeline.

[0010] In a third aspect, the present invention proposes the use of the above-described engineered strain in the preparation of a medicament for improving or treating obesity.

[0011] In a fourth aspect, the present invention proposes the use of the above-mentioned engineered strain in the preparation of a medicament for improving or treating metabolic disorders, including type 2 diabetes, fatty liver, and cardiovascular metabolic abnormalities.

[0012] According to the application of the present invention, the above-mentioned engineered strains can achieve sustained local intestinal delivery via oral administration, providing a more gradual and effective exposure, which helps to reduce the risk of gastrointestinal adverse reactions caused by excessively high peak concentrations. In a mouse model induced by a high-fat diet via gavage, the above-mentioned engineered strains exhibited sustained weight loss, accompanied by a significant decrease in blood glucose levels and a marked improvement in insulin sensitivity, indicating that engineered microorganism-mediated dual agonist delivery can significantly improve overall metabolic homeostasis. Therefore, by constructing engineered strains with a moduloable GLP-1 / GIP expression ratio, a novel approach is provided to overcome the limitations of existing biological agent delivery methods, potentially improving treatment accessibility and long-term adherence, and opening new avenues for the sustainable translational application of metabolic diseases.

[0013] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0014] Figure 1 A schematic diagram illustrating the principle of engineered Saccharomyces boulardii secreting polypeptides and releasing GLP-1 / GIPs via Kex1 / Kex2 / Ste13 processing according to an embodiment of the present invention; Figure 2 Figure (A) shows the gel electrophoresis verification results (A) and the gradient dilution engineered bacteria concentration estimation results (B) of the single-strain integration ratio adjustable GLP-1:GIP expression module according to an embodiment of the present invention. Figure 3The figure shows the improvement results of single-strain adjustable GLP-1:GIP co-delivery on body weight, OGTT, and ITT in obese model mice according to embodiments of the present invention; where A is a schematic diagram of the experimental design of single strains expressing GLP-1 and GIP at a defined copy number ratio; B is the change in body weight (absolute value and percentage) during treatment; C is the oral glucose tolerance test (OGTT) curve; D is the insulin tolerance test (ITT) curve; E is the comparison of the area under the curve (AUC) of blood glucose levels at different time points during OGTT between groups; F is the comparison of the area under the curve (AUC) of blood glucose levels at different time points during ITT between groups. Detailed Implementation

[0015] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0016] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0017] The test materials used in this invention are all commercially available products and can be purchased on the market; unless otherwise specified, the experiments involved are all conventional experimental methods.

[0018] Materials used: Wild-type *Saccharomyces boulardii* (strain model: CNCM I-745) and TOP10 strains were commercially available. The wild-type *Saccharomyces boulardii* was used to knock out ura3 to construct a uracil-deficient *Saccharomyces boulardii* chassis strain, which was then used for engineering modification. TOP10 strains were used for vector construction. The *Pichia pastoris* expression vector pGAPZ(alpha)A was purchased from Thermo Fisher Scientific, and the PBase transposase expression vector was obtained from a published paper (Biotechnology Journal, 2024, 19:e202400527). Phusion high-fidelity DNA polymerase and restriction endonucleases were purchased from Xiamen Lulong Biotechnology Development Co., Ltd. Plasmid extraction kits, DNA purification kits, gel extraction kits, and genomic DNA extraction kits were purchased from Hangzhou Borui Biotechnology.

[0019] The composition of LB medium is: 10 g·L -1 Tryptone, 5 g·L -1 Yeast extract, 10 g·L -1 Add NaCl and double-distilled water to a final volume of 1 L, then sterilize at 121°C and 0.1 MPa pressure for 20 min.

[0020] The YPD culture medium composition is: 10 g·L -1 Yeast extract, 20 g·L -1 Peptone, 20 g·L -1 Add glucose and double-distilled water to a final volume of 1 L, then sterilize at 121°C and 0.1 MPa pressure for 20 min.

[0021] The YNBD-URA medium composition is: 6.7 g·L⁻¹ -1 Yeast nitrogen source basal, 1.4 g·L -1 Yeast auxotrophic culture medium supplement (uridine-free), 20 g·L -1 Add D-glucose and double-distilled water to a final volume of 1 L, then sterilize at 121°C and 0.1 MPa for 15 min.

[0022] Table 1 Primers used for PCR amplification

[0023] Table 2 DNA sequences of the expression module

[0024] Continued from Table 2: DNA sequence of the expression module

[0025] Continued from Table 2: DNA sequence of the expression module

[0026] Saccharomyces boulardii, as a eukaryotic probiotic, possesses characteristics such as tolerance to gastric acid and bile salts, short-term survival in the intestine, and regulation of the intestinal barrier and immune microenvironment. Furthermore, it has eukaryotic secretion and endogenous protease processing capabilities, making it suitable for the biosynthesis and secretion of polypeptide therapeutic molecules. For example... Figure 1As shown, this application utilizes engineered *Saccharomyces boulardii* to express the GLP-1 / GIP dual agonist as a "polypeptide precursor" within yeast. Leveraging the yeast's endogenous secretion system and protease processing system, the precise release of the mature active peptide is achieved. Ultimately, oral administration allows the yeast to continuously deliver the dual agonist in the intestine, exerting a metabolic regulatory effect. Specifically, the genome of the engineered *Saccharomyces boulardii* integrates a polypeptide coding sequence containing an MFαSP secretion signal peptide, a GLP-1 repeat unit, a linker sequence, and a GIP repeat unit. Ribosomes bind to this coding sequence to initiate translation, synthesizing a polypeptide precursor containing the MFαSP signal peptide (with MFαSP at the N-terminus, GLP-1 and GIP repeat units tandemly in the middle, and KR dibasic sites and EAEAEA acidic spacer peptides between the repeat units). Signal recognition particles (SRPs) bind to MFαSP, guiding the ribosome-peptide complex to the Sec secretion complex on the cell membrane, allowing the polypeptide precursor to enter the endoplasmic reticulum through the Sec complex and initiate the secretion pathway. After initial folding in the endoplasmic reticulum, the polypeptide precursor is transported to the Golgi apparatus. With the synergistic action of three endogenous yeast proteases, mature GLP-1 and GIP are released. (Kex2 protease: recognizes and cleaves the KR dibasic site (Lys-Arg) between repeat units, initially cleaving the polypeptide precursor into GLP-1 monomer fragments, GIP monomer fragments, and intermediate products containing acidic spacer peptides; Ste13 protease: recognizes and cleaves the acidic spacer peptide sequence (EAEAEA), removing residual spacer peptide fragments and ensuring the amino acid sequence integrity of GLP-1 and GIP; Kex1 protease: further prunes the residual terminal amino acids after cleavage, enhancing the biological activity of the mature peptide). Mature GLP-1 agonists (containing A8G / K34R mutations, resistant to DPP-4 degradation) and GIP agonists, processed by the Golgi apparatus, are secreted extracellularly via yeast exocytosis. After oral administration, *Saccharomyces boulardii*, with its natural tolerance to gastric acid and bile salts, survives and colonizes the intestinal mucosa, continuously secreting both agonists into the intestinal tract. GLP-1 and GIP in the intestine bind to GLP-1 and GIP receptors on the surface of intestinal epithelial cells, respectively, synergistically suppressing appetite, promoting insulin secretion, and improving insulin sensitivity, ultimately achieving weight loss and blood glucose homeostasis regulation. The copy number ratio of GLP-1 to GIP repeat units in the polypeptide coding sequence (e.g., 2:2, 4:2, 6:2) can be precisely controlled. Combined with efficient cleavage of the linker sequence by yeast endogenous proteases, this ensures that the proportion of both agonists secreted into the intestine matches the designed ratio, avoiding ratio drift caused by mixed delivery from multiple strains, and achieving programmable synergistic optimization of "configuration-dosage-efficacy".

[0027] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0028] Example 1: Construction of a multipeptide expression module for secreting GLP-1 agonists To improve the stability of GLP-1 in vivo, especially in the intestinal environment, and to prolong its effective duration of action, this embodiment optimized the structure of the GLP-1 peptide to obtain a GLP-1 agonist sequence with DPP-4 resistance characteristics. Figure 1 As shown, in the natural GLP-1 molecule (amino acid sequence as shown in SEQ ID No. 15: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG), the peptide bond between the 8th and 9th amino acids is the main recognition and cleavage region of dipeptidyl peptidase-4 (DPP-4), resulting in a short half-life of natural GLP-1 in vivo. Therefore, this embodiment aims to improve DPP-4 resistance and maintain receptor agonist activity by substituting amino acids at key sites in GLP-1.

[0029] Specifically, the 8th amino acid of GLP-1 is replaced with glycine (Gly), denoted as A8G substitution; further, the 34th amino acid is replaced with arginine (Arg), denoted as K34R substitution. The GLP-1 analog obtained through these site substitutions avoids DPP-4 cleavage and degradation without significantly affecting its binding to and agonistic ability with the GLP-1 receptor.

[0030] Based on the amino acid sequence of the GLP-1 analog (HGEGTFTSDVSSYLEGQAAKEFIAWLVRGRG, SEQ ID No. 16), nucleotide sequence design and codon optimization were performed to obtain the coding sequence corresponding to the GLP-1 analog, such as the coding sequence shown in SEQ ID No. 10, which was obtained by GenScript (Nanjing, Jiangsu) through chemical synthesis.

[0031] Example 2: Construction of a multipeptide expression module for secreting GIP agonists To achieve efficient biosynthesis and secretory processing of GIP peptides in engineered probiotic yeast, this embodiment provides a GIP synthesis and construction strategy that uses multiple copy tandem and yeast endogenous protease cleavage sites to achieve the release of mature GIP.

[0032] The natural mature peptide sequence of glucose-dependent insulinotropic peptide (GIP) (YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ, SEQ ID No. 17) was selected as the target sequence and used as the basic structure of GIP receptor agonist.

[0033] A dual-basic site KR (Lys-Arg) that can be recognized and cleaved by yeast endogenous proteases is set between adjacent GIP repeat units, so that the precursor peptide can be cleaved by Kex1 and / or Kex2 during Golgi processing, releasing the mature GIP peptide.

[0034] The nucleotide coding sequence of the GIP(2x) precursor polypeptide was designed based on its structure and codons were optimized for yeast expression, as shown in the coding sequence SEQ ID No. 11, which was obtained by chemical synthesis by GenScript (Nanjing, Jiangsu).

[0035] Example 3: Construction of a single-strain GLP-1 / GIP ratio adjustable co-delivery expression module To achieve proportional co-delivery within a single strain, GLP-1 and the GIP module are encoded in the same polypeptide precursor and linked by an acidic spacer peptide sequence that can be cleaved by Ste13. The acidic spacer peptide sequence is EAEAEA.

[0036] Construct expression modules with different GLP-1:GIP copy number ratios, including but not limited to: GLP-1(2x)-GIP(2x), GLP-1(4x)-GIP(2x), and GLP-1(6x)-GIP(2x), to achieve programmable control of different output ratios and effect intensities.

[0037] Specifically, using the pGAPZ(alpha)A vector as a template, and with MFα_BsaI_fwd (nucleotide sequence SEQ ID No. 1) and MFα_BsaI_rev (nucleotide sequence SEQ ID No. 2) as primers, the MFα fragment was amplified. After digestion with BamHI and SalI, the fragment was inserted into the BamHI and XhoI sites of the pUC18BP-URA-TEF2 vector (disclosed in Biotechnology Journal, 2024, 19:e202400527) to construct the pUC18BP-URA-TEF2-MFαSP vector. Conditions: 98℃ for 2 min, 98℃ for 10 s, 56℃ for 10 s, 72℃ for 1 min, 30 cycles; 72℃ for 2 min.

[0038] Using pKan-GLP-1(6x) synthesized by GenScript (Nanjing, Jiangsu) as a template, the nucleotide sequence of GLP-1(6x) is shown in SEQ ID No. 10. Using GLP-1_XhoI_fwd (nucleotide sequence SEQ ID No. 4) and GLP-1_OE2x_rev (nucleotide sequence SEQ ID No. 5) as primers (Table 1), the GLP-1(2x)-OE fragment was obtained by PCR amplification. Using pKan-GIP(2x) synthesized by GenScript (Nanjing, Jiangsu) as a template, the nucleotide sequence of GIP(2x) is shown in SEQ ID No. 11. Using GIP_OE2x_fwd (nucleotide sequence SEQ ID No. 8) and GIP_XbaI_rev (nucleotide sequence SEQ ID No. 9) as primers, the GIP(2x)-OE fragment was obtained by PCR amplification. Using GLP-1(2x)-OE and GIP(2x)-OE fragments as templates, and with GLP-1_XhoI_fwd (nucleotide sequence SEQ ID No. 4) and GIP_XbaI_rev (nucleotide sequence SEQ ID No. 9) as primers, GLP-1(2x)-GIP(2x) was amplified by PCR. The recombinant plasmid pUC18BP-URA-TEF2-MFαSP was constructed by inserting it into the pUC18BP-URA-TEF2-MFαSP vector through the XhoI and XbaI sites. The nucleotide sequence of this recombinant plasmid is shown in SEQ ID No. 12. The PCR amplification conditions were: 98℃ for 2 min, 98℃ for 10 s, 56℃ for 10 s, 72℃ for 1 min, for 30 cycles; 72℃ for 2 min.

[0039] Using pKan-GLP-1(6x) as a template, and with primers GLP-1_XhoI_fwd (SEQ ID No. 4) and GLP-1_OE4x_rev (SEQ ID No. 6) as primers, the GLP-1(4x)-OE fragment was amplified by PCR. Using the GLP-1(4x)-OE fragment and the aforementioned GIP(2x)-OE fragment as templates, and with primers GLP-1_XhoI_fwd (SEQ ID No. 4) and GIP_XbaI_rev (SEQ ID No. 9) as primers, the GLP-1(4x)-GIP(2x) fragment was amplified by PCR. The recombinant plasmid pUC18BP-URA-TEF2-MFαSP was constructed by inserting it into the pUC18BP-URA-TEF2-MFαSP vector through the XhoI and XbaI sites. The nucleotide sequence of the recombinant plasmid is shown in SEQ ID No. 13. The PCR amplification conditions were as follows: 98℃ for 2 min, 98℃ for 10 s, 56℃ for 10 s, 72℃ for 1 min, for 30 cycles; 72℃ for 2 min.

[0040] Using pKan-GLP-1(6x) as a template, and with primers GLP-1_XhoI_fwd (SEQ ID No. 4) and GLP-1_OE6x_rev (SEQ ID No. 7) as primers, the GLP-1(6x)-OE fragment was amplified by PCR. Using GLP-1(6x)-OE and the aforementioned GIP(2x)-OE as templates, and with primers GLP-1_XhoI_fwd (SEQ ID No. 4) and GIP_XbaI_rev (SEQ ID No. 9) as primers, the GLP-1(6x)-GIP(2x) fragment was amplified by PCR. The recombinant plasmid pUC18BP-URA-TEF2-MFαSP was constructed by inserting it into the pUC18BP-URA-TEF2-MFαSP vector through the XhoI and XbaI sites. The nucleotide sequence of the recombinant plasmid is shown in SEQ ID No. 14. The PCR amplification conditions were as follows: 98℃ for 2 min, 98℃ for 10 s, 56℃ for 10 s, 72℃ for 1 min, for 30 cycles; 72℃ for 2 min.

[0041] All recombinant plasmids were verified using the nucleotide sequence PB5_verify_fwd shown in SEQ ID No. 3 and the nucleotide sequence GIP_Xba1_rev shown in SEQ ID No. 9, and sequencing was performed to ensure that the results were completely consistent with the designed plasmid DNA sequence, thus obtaining the correct recombinant plasmid.

[0042] Example 4: Genome integration of expression modules using the PiggyBac system Uracil-deficient *Saccharomyces boulardii*: Using the genome of *Saccharomyces cerevisiae* strain BY4741 as a template (purchased from the American Collection of Saccharomyces Culture Collection, Catalog No. 4040002), the ura3-del homologous knockout fragment was amplified using ura3_F500 (GATGCTAAGAGATAGTGATG, SEQ ID No. 18) and ura3_R500 (CATGCGTTTGTACTCTAATC, SEQ ID No. 19) as primers. After purification and recovery by gel electrophoresis, the fragment was mixed with wild-type *Saccharomyces boulardii* electrotransformation competent cells. After electrotransformation, the fragments were plated on YNBD + 1 g / L 5-fluorouracil solid agar plates and cultured for 3-5 days. The fragments were repeatedly streaked on YNBD + 1 g / L 5-fluorouracil solid agar plates for separation. The resulting single colonies were then enriched on YNBD uracil-deficient medium and cultured for 3-5 days, exhibiting a no-growth phenotype. The knockout was confirmed by PCR.

[0043] The donor vector containing the "selection marker URA-TEF2p-MFαSP-polypeptide coding sequence-terminator" was co-introduced into uracil-deficient Saccharomyces boulardii with the PBase transposase expression vector to obtain recombinant Saccharomyces boulardii.

[0044] The recombinant *Saccharomyces boulardii* was screened for positive clones in uracil-deficient medium YNBD-URA. All positive clones were lysed in 40 mmol / L sodium hydroxide solution at 98°C for 45 min. Amplification was then performed using primers for GLP-1_XhoI_fwd (SEQ ID No. 4) and GIP_XbaI_rev (SEQ ID No. 9). Gel electrophoresis confirmed successful integration of the multipeptide gene. Figure 2 A). Subsequently, after three rounds of subculturing in YPD liquid medium, the cells were streaked to YPD solid medium. The grown single clones were confirmed by transposase-specific primers to have no amplification bands, ensuring the loss of the PBase vector, and thus obtaining stable genome-integrated engineered dual-agonist Saccharomyces boulardii strains: S. boulardii::GLP-1(2x)-GIP(2x), S. boulardii::GLP-1(4x)-GIP(2x), and S. boulardii::GLP-1(6x)-GIP(2x).

[0045] Example 5: Effect of engineered dual-agonist Saccharomyces boulardii strain on the improvement of a high-fat diet-induced obesity model. like Figure 3As shown in Figure A, 6-8 week old male C57BL / 6J mice were randomly divided into a blank control group, a positive control group (semaglutide injection group), and three engineered bacterial strain groups: S. boulardii::GLP-1(2x)-GIP(2x), S. boulardii::GLP-1(4x)-GIP(2x), and S. boulardii::GLP-1(6x)-GIP(2x). Except for the blank control group, which received a standard diet, the other groups were given a high-fat diet to induce an obesity model. After successful model induction, the engineered bacterial strain groups were fed approximately 10 7 CFU / dose (reference) Figure 2 B) Administer orally via gavage for 30 consecutive days.

[0046] Weight changes were recorded during the intervention, and oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed at the end of the administration period to detect changes in blood glucose.

[0047] The results are as follows Figure 3 As shown in B to 3F, compared with the control group, the body weight of mice in all three engineered strain groups was significantly reduced, and the OGTT and ITT indices were significantly improved. Different GLP-1:GIP ratio strains showed a proportional-dependent trend in terms of body weight and fat improvement. The comprehensive metabolic benefit of strains with a high GLP-1 ratio was more significant, that is, the comprehensive metabolic benefit of the S. boulardii::GLP-1(6x)-GIP(2x) group was the most significant. This indicates that the engineered strains constructed in this application can effectively exert the synergistic therapeutic effect of dual agonists and achieve programmable synergistic optimization of "configuration-dosage-efficacy".

[0048] In summary, the *Saccharomyces boulardii* strain constructed in this invention for intestinal delivery of GLP-1 / GIP dual receptor agonists achieves sustained intestinal delivery of the dual agonists via oral administration, overcoming the core pain points of existing technologies such as injection dependence, low bioavailability, and severe adverse reactions. The fermentation process of this strain is mature, allowing for large-scale production at low cost. The resulting drugs, functional foods, or dietary supplements offer advantages such as convenient oral administration, high compliance, and good safety, making them widely applicable in the treatment of metabolic diseases such as obesity, type 2 diabetes, and fatty liver, and possessing broad industrial application prospects and market value.

[0049] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0050] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An engineered bacterial strain for intestinal delivery of a GLP-1 / GIP dual receptor agonist, characterized in that, The engineered strain uses uracil-deficient *Bacillus boulardii* as the substrate strain. The genome of the substrate strain integrates a polypeptide expression module. The polypeptide expression module includes a secretion signal peptide coding sequence and a polypeptide coding sequence fused with the secretion signal peptide coding sequence. The polypeptide coding sequence encodes a polypeptide formed by multiple glucagon-like peptide-1 repeat units and multiple glucose-dependent insulinotropic peptide repeat units tandemly. Restriction sites that can be cleaved by yeast endogenous proteases are set between adjacent repeat units so that the polypeptide can be cleaved in the Golgi apparatus or during secretory processing, releasing mature GLP-1 agonists and GIP agonists. The glucagon-like peptide-1 repeat unit is a dipeptidyl peptidase-4 resistant GLP-1 analog, which is a variant of wild-type GLP-1 obtained by modifying it, wherein the modification includes replacing the 8th amino acid with glycine and replacing the 34th amino acid with arginine. The amino acid sequence of the glucose-dependent insulinotropic polypeptide repeat unit is shown in SEQ ID No.

17.

2. The engineered strain as described in claim 1, characterized in that, The ratio of glucagon-like peptide-1 repeat units to glucose-dependent insulinotropic peptide repeat units in the polypeptide is (1-4):

1.

3. The engineered strain as described in claim 1, characterized in that, The cleavage sites between adjacent repeat units are KR dibasic sites, which can be recognized and cleaved by yeast endogenous Kex1 and / or Kex2 proteases.

4. The engineered strain as described in claim 1, characterized in that, The polypeptide further includes a spacer peptide sequence that connects adjacent glucagon-like peptide-1 repeat units and glucose-dependent insulinotropic peptide repeat units in the polypeptide, wherein the spacer peptide is an acidic spacer peptide sequence that can be cleaved by the Ste13 protease.

5. The engineered strain as described in claim 4, characterized in that, The acidic spacer peptide sequence is EAEAEA.

6. A method for preparing the engineered strain according to any one of claims 1-5, characterized in that, Includes the following steps: Step 1: Construct a donor vector containing the polypeptide expression module; Step 2: The donor vector and the PBase transposase expression vector are jointly introduced into the chassis strain to obtain the recombinant strain; Step 3: Screen positive clones of the recombinant strain on uracil-deficient medium. After passage, the PBase transposase expression vector is removed from the positive clones to obtain stable integrated engineered strains.

7. The method as described in claim 6, characterized in that, Step 1 includes: Using pKan-GLP-1(6x) as a template, and with primers GLP-1_XhoI_fwd (SEQ ID No. 4) and GLP-1_OE2x_rev (SEQ ID No. 5) as primers, the GLP-1(2x)-OE fragment was amplified by PCR. Using pKan-GIP(2x) as a template, and with primers GIP_OE2x_fwd (SEQ ID No. 8) and GIP_XbaI_rev (SEQ ID No. 9) as primers, the GIP(2x)-OE fragment was amplified by PCR. Using the GLP-1(2x)-OE fragment and the GIP(2x)-OE fragment as templates, and with primers GLP-1_XhoI_fwd (SEQ ID No. 4) and GLP-1_OE2x_rev (SEQ ID No. 5) as primers, the GLP-1(6x)-OE fragment was amplified by PCR. Using GIP_XbaI_rev as primer No. 9, GLP-1(2x)-GIP(2x) was amplified by PCR; the recombinant plasmid pUC18BP-URA-TEF2-MFαSP was constructed by inserting XhoI and XbaI sites into the pUC18BP-URA-TEF2-MFαSP vector, and the nucleotide sequence shown in SEQ ID No. 12 was constructed. Alternatively, using pKan-GLP-1(6x) as a template, and with GLP-1_XhoI_fwd (nucleotide sequence SEQ ID No. 4) and GLP-1_OE4x_rev (nucleotide sequence SEQ ID No. 6) as primers, PCR amplification was performed to obtain the GLP-1(4x)-OE fragment; using the GLP-1(4x)-OE fragment and the aforementioned GIP(2x)-OE fragment as templates, and with GLP-1_XhoI_fwd (nucleotide sequence SEQ ID No. 4) and GIP_XbaI_rev (nucleotide sequence SEQ ID No. 9) as primers, PCR amplification was performed to obtain GLP-1(4x)-GIP(2x); the recombinant plasmid pUC18BP-URA-TEF2-MFαSP with the nucleotide sequence shown in SEQ ID No. 13 was constructed by inserting the XhoI and XbaI sites into the pUC18BP-URA-TEF2-MFαSP vector; Alternatively, using pKan-GLP-1(6x) as a template, and with GLP-1_XhoI_fwd (nucleotide sequence SEQ ID No. 4) and GLP-1_OE6x_rev (nucleotide sequence SEQ ID No. 7) as primers, the GLP-1(6x)-OE fragment was amplified by PCR. Using GLP-1(6x)-OE and the aforementioned GIP(2x)-OE as templates, and with GLP-1_XhoI_fwd (nucleotide sequence SEQ ID No. 4) and GIP_XbaI_rev (nucleotide sequence SEQ ID No. 9) as primers, GLP-1(6x)-GIP(2x) was amplified by PCR. The recombinant plasmid pUC18BP-URA-TEF2-MFαSP, with the nucleotide sequence shown in SEQ ID No. 14, was constructed by inserting XhoI and XbaI sites into the pUC18BP-URA-TEF2-MFαSP vector. The GLP-1(6x) nucleotide sequence is shown in SEQ ID No.

10.

8. The use of the engineered strain according to any one of claims 1-5 in the preparation of a medicament for improving or treating obesity.

9. The use of the engineered strain according to any one of claims 1-5 in the preparation of a medicament for improving or treating a metabolic disorder, wherein the metabolic disorder is type 2 diabetes.

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