Engineered probiotics for preventing or treating alzheimer's disease and use thereof

Abstract

The present application provides a yeast strain capable of producing 5-methyltetrahydrofolate, a construction method and a fermentation method thereof, and the use of the strain or its fermentation product in the preparation of 5-methyltetrahydrofolate or its salt, in the supplementation of 5-methyltetrahydrofolate or its salt in a subject. Further, the present application also provides the use of the strain or its fermentation product in the prevention and / or treatment of Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and microbial fermentation technology, specifically relating to a yeast strain capable of producing 5-methyltetrahydrofolate, its construction method and fermentation method, and the use of this strain or its fermentation product in the preparation of 5-methyltetrahydrofolate or its salts, and in supplementing subjects with 5-methyltetrahydrofolate or its salts. Furthermore, this invention also relates to the application of this strain or its fermentation product in the prevention and / or treatment of Alzheimer's disease. Background Technology

[0002] 5-Methyltetrahydrofolate (5-MTHF) is the active form of folic acid in the human body, naturally found in leafy green vegetables and animal liver, but in low amounts. Compared to regular folic acid, 5-MTHF can be directly utilized by cells without the metabolic conversion by dihydrofolate reductase (DHFR) and methylenetetrahydrofolate reductase (MTHFR), thus significantly improving bioavailability (by approximately 30-50%), making it particularly suitable for individuals with MTHFR gene mutations. Its physiological functions include participation in DNA synthesis and repair, support for erythropoiesis, and efficient regulation of homocysteine ​​metabolism, thus offering greater clinical advantages in reducing cardiovascular risk and preventing neural tube defects in fetuses.

[0003] The key differences between 5-MTHF and regular folic acid lie in several aspects: In terms of metabolic pathways, regular folic acid requires multiple conversions via DHFR and MTHFR to be utilized, while 5-MTHF can directly enter the methyl cycle, avoiding metabolic activation. Regarding bioavailability, regular folic acid is lower due to limitations imposed by intestinal absorption and enzyme activity, while 5-MTHF is unaffected by MTHFR gene mutations and is more efficient. In terms of target populations, regular folic acid is suitable for the general population, while 5-MTHF is more suitable for individuals with MTHFR mutations, the elderly, and patients with digestive disorders. From a safety perspective, excessive intake of regular folic acid may lead to the accumulation of unmetabolized folic acid, while 5-MTHF, due to its efficient metabolic pathway, carries no risk of accumulation and is more advantageous in preventing disease and lowering homocysteine ​​levels.

[0004] Saccharomyces boulardii is an acid- and bile-resistant, non-pathogenic probiotic yeast widely used to prevent and treat diarrhea and regulate gut microbiota. Its unique anti-inflammatory, anti-pathogen, and gut barrier-enhancing properties make it an ideal carrier for microbial therapy. Summary of the Invention

[0005] Based on extensive experimental research, the inventors of this application have discovered that specific gene modifications can endow engineered strains containing such gene modifications with the ability to synthesize active folic acid (5-methyltetrahydrofolate), and different gene modifications can result in significant differences in the 5-methyltetrahydrofolate synthesis capacity of the engineered strains. The engineered strains producing 5-methyltetrahydrofolate based on this application will greatly reduce the production cost of 5-methyltetrahydrofolate, simplify the process, and promote the green and clean production of high-value pharmaceutical and biochemical products, possessing significant economic and social value.

[0006] Furthermore, the inventors of this application have unexpectedly discovered that the engineered strain containing specific gene modifications provided in this application can be significantly beneficial for the prevention and / or treatment of Alzheimer's disease, and experiments have confirmed that it can produce significant therapeutic effects in various Alzheimer's disease mouse models.

[0007] Therefore, in a first aspect, this application provides engineered bacteria comprising one or more gene modifications selected from:

[0008] (a) Gene modifications that enhance the expression of the FOL3 gene;

[0009] (b) Gene modifications that enhance the expression of the DFR1 gene;

[0010] (c) Gene modifications that enhance the expression of the ERC1 gene;

[0011] (d) Gene modifications that enhance the expression of the MET13 gene.

[0012] As used herein, the genes FOL3 (encoding dihydrofolate synthase), DFR1 (encoding dihydrofolate reductase), ERC1 (encoding ethionine resistance conferring protein 1), and MET13 (encoding methylenetetrahydrofolate reductase) have the meanings commonly understood by those skilled in the art, and their structural and sequence information is available from various public databases. Obtaining the structural and sequence information of these genes is entirely within the capabilities of those skilled in the art.

[0013] In some embodiments, the engineered bacteria comprises:

[0014] (1) The modification (a);

[0015] (2) The modifications (a) and (b);

[0016] (3) The modifications (a), (b) and (c); or,

[0017] (4) Modifications (a), (b), (c) and (d).

[0018] In some embodiments, the gene modifications that enhance gene expression described in (a) to (d) are each independently selected from: promoters that enhance gene expression, gene linkage or introduction into enhancers, gene copy number increase, and any combination thereof.

[0019] In some embodiments, the gene modification that enhances gene expression described in (a) to (d) is to increase the copy number of the gene.

[0020] It will be readily understood by those skilled in the art that increasing the copy number of a gene does not mean that each copy of the gene must have an identical nucleotide sequence, as long as each copy can perform its original functional activity (e.g., achieve protein expression).

[0021] In a second aspect, this application provides an engineered bacterium containing one or more exogenously introduced genes, said genes being selected from: MET13, DFR1, FOL3, ERC1, and any combination thereof.

[0022] In some embodiments, the engineered bacteria comprise exogenously introduced:

[0023] (i) Gene FOL3;

[0024] (ii) Genes FOL3 and DFR1;

[0025] (iii) Genes DFR1, FOL3, and ERC1; or,

[0026] (iv) Genes MET13, DFR1, FOL3 and ERC1.

[0027] In some embodiments, the engineered bacteria contains an exogenously introduced gene FOL3 operatively linked to a TDH3 promoter, for example, expressed by the TDH3 promoter. In some embodiments, the exogenously introduced gene FOL3 is operatively linked to a TEF1 terminator (for example, transcription termination regulated by the TEF1 terminator).

[0028] In some embodiments, the engineered bacteria contains an exogenously introduced gene DFR1 operatively linked to a TEF1 promoter, for example, expressed by the TEF1 promoter. In some embodiments, the exogenously introduced gene DFR1 is operatively linked to a TDH3 terminator (for example, transcription termination regulated by the TDH3 terminator).

[0029] In some embodiments, the engineered bacteria contains an exogenously introduced gene ERC1 operatively linked to a PGK1 promoter, for example, expressed by the PGK1 promoter. In some embodiments, the exogenously introduced gene ERC1 is operatively linked to a PGK1 terminator (e.g., transcription termination regulated by the PGK1 terminator).

[0030] In some embodiments, the engineered bacteria contains an exogenously introduced gene MET13 operatively linked to a TPI1 promoter, for example, expressed by the TPI1 promoter. In some embodiments, the exogenously introduced gene MET13 is operatively linked to an ADH1 terminator (for example, transcription termination regulated by the ADH1 terminator).

[0031] In some embodiments, the codon composition of the nucleotide sequence of the one or more exogenously introduced genes is adapted to the codon bias of the genes of the engineered bacteria. In some embodiments, the codon composition of the nucleotide sequence of the one or more exogenously introduced genes is adapted to the codon bias of the Saccharomyces boulardii (e.g., Saccharomyces boulardii MYA-796) gene.

[0032] In some embodiments, one or more of the exogenously introduced genes are present in the genome of the engineered bacteria.

[0033] In some embodiments, the one or more genes are introduced into the non-coding region of the engineered bacterial genome, for example, without affecting the expression / regulation of endogenous genes in the engineered bacteria. For example, the insertion sites of the one or more genes in the genome of the engineered bacteria satisfy the following:

[0034] (a) Located outside the transcriptional unit of a protein-coding gene;

[0035] (b) at least 50 kb from the transcription start site of any endogenous gene; and / or,

[0036] (c) Not located inside any known microRNA (miRNA) or long noncoding RNA (lncRNA) gene.

[0037] One or more genes can be introduced into the genome of the engineered bacteria using various methods conventionally used in the art (e.g., genetic engineering techniques), ensuring that the introduced genes can perform their original functional activities (e.g., achieve protein expression). Based on the disclosure of this application, the selection and / or implementation of such methods are entirely within the capabilities of those skilled in the art.

[0038] In some embodiments, the exogenously introduced genes MET13, DFR1, FOL3, and / or ERC1 each have one or more independent copies. It will be readily understood by those skilled in the art that it is not required that the individual copies of the genes have identical nucleotide sequences, only that each copy can perform its original functional activity (e.g., achieve protein expression).

[0039] In some embodiments, the exogenously introduced genes MET13, DFR1, FOL3 and / or ERC1 are derived from Saccharomyces cerevisiae or Saccharomyces boulardii.

[0040] In some embodiments, the exogenously introduced genes MET13, DFR1, FOL3 and / or ERC1 are derived from Saccharomyces cerevisiae, for example, Saccharomyces cerevisiae S288C.

[0041] Those skilled in the art will understand that *Saccharomyces cerevisiae* and *Saccharomyces boulardii* share high homology, and their genes (e.g., MET13, DFR1, FOL3, ERC1) exhibit high sequence identity and adaptability. Therefore, the exogenous genes MET13, DFR1, FOL3, and / or ERC1 introduced into the engineered strain can theoretically also originate from *Saccharomyces boulardii*. Thus, in some embodiments, the exogenously introduced genes MET13, DFR1, FOL3, and / or ERC1 are derived from *Saccharomyces boulardii*, for example, *Saccharomyces boulardii* MYA-796.

[0042] Those skilled in the art will know that Saccharomyces cerevisiae S288C and Saccharomyces boulardii MYA-796 are both widely used yeast strains in the field and can be obtained through various conventional means, such as purchasing them from the American Type Culture Collection (ATCC).

[0043] In some embodiments of the first or second aspect described above, the engineered bacteria is yeast.

[0044] In some embodiments of the first or second aspect above, the engineered bacteria are selected from: Saccharomyces boulardii, Saccharomyces pastorianus, Pichiastipitis, Saccharomyces Bayanus, and Candida shehatae.

[0045] Due to its application safety, natural acid resistance, and excellent intestinal colonization ability, *Saccharomyces boulardii* can serve as the dominant substrate bacteria for the engineered strains of this application, and can be developed into an oral "living folic acid factory" to synthesize active folic acid (5-methyltetrahydrofolate) in situ within the intestine. Therefore, in some embodiments of the first or second aspect above, the engineered strain is *Saccharomyces boulardii*, for example, *Saccharomyces boulardii* MYA-796.

[0046] In some embodiments of the first aspect described above, the engineered bacteria are obtained from Saccharomyces boulardii (e.g., Saccharomyces boulardii MYA-796) through the aforementioned gene modification.

[0047] In some embodiments of the second aspect above, the engineered bacteria are obtained by exogenously introducing one or more genes from Saccharomyces boulardii (e.g., Saccharomyces boulardii MYA-796).

[0048] In some embodiments of the first or second aspect above, the 5-methyltetrahydrofolate synthesis capacity of the engineered bacteria is at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 6 times, at least 6.5 times, at least 7 times, at least 7.5 times, or at least 8 times that of its corresponding chassis bacteria.

[0049] In a third aspect, this application provides a method for constructing the engineered bacteria of the first or second aspect, comprising:

[0050] (1) Provide chassis bacteria; and,

[0051] (2) Introduce the gene modification defined in the first aspect into the sclerotium, or introduce one or more genes exogenously into the sclerotium, the genes being selected from: MET13, DFR1, FOL3, ERC1, and any combination thereof.

[0052] Preferably, step (2) includes: introducing one or more genes exogenously into the chassis bacteria, wherein the genes are selected from: MET13, DFR1, FOL3, ERC1, and any combination thereof.

[0053] In some implementations, step (2) includes the exogenous introduction of the chassis bacteria:

[0054] (i) Gene FOL3;

[0055] (ii) Genes FOL3 and DFR1;

[0056] (iii) Genes DFR1, FOL3, and ERC1; or,

[0057] (iv) Genes MET13, DFR1, FOL3 and ERC1.

[0058] In some embodiments, the method includes the exogenous introduction of the gene FOL3 into the *Bacterium tumefaciens*, which is operatively linked to a TDH3 promoter, for example, expressed by the TDH3 promoter. In some embodiments, the exogenously introduced gene FOL3 is operatively linked to a TEF1 terminator (for example, transcription termination regulated by the TEF1 terminator).

[0059] In some embodiments, the method includes the exogenous introduction of a gene DFR1 into the *Chaetomium*, which is operatively linked to a TEF1 promoter, for example, expressed by the TEF1 promoter. In some embodiments, the exogenously introduced gene DFR1 is operatively linked to a TDH3 terminator (for example, transcription termination regulated by the TDH3 terminator).

[0060] In some embodiments, the method includes the exogenous introduction of a gene ERC1 into the *Chaetomium*, which is operatively linked to a PGK1 promoter, for example, expressed by the PGK1 promoter. In some embodiments, the exogenously introduced gene ERC1 is operatively linked to a PGK1 terminator (for example, transcription termination regulated by the PGK1 terminator).

[0061] In some embodiments, the method includes the exogenous introduction of the gene MET13 into the *Chaetomium*, which is operatively linked to a TPI1 promoter, for example, expressed by the TPI1 promoter. In some embodiments, the exogenously introduced gene MET13 is operatively linked to an ADH1 terminator (for example, transcription termination regulated by the ADH1 terminator).

[0062] In some embodiments, the codon composition of the nucleotide sequence of the one or more exogenously introduced genes is adapted to the codon bias of the genes of the engineered bacteria. In some embodiments, the codon composition of the nucleotide sequence of the one or more exogenously introduced genes is adapted to the codon bias of the Saccharomyces boulardii (e.g., Saccharomyces boulardii MYA-796) gene.

[0063] In some embodiments, one or more genes are introduced into the genome of the engineered bacteria.

[0064] In some embodiments, the one or more genes are introduced into the non-coding region of the engineered bacterial genome, for example, without affecting the expression / regulation of endogenous genes in the engineered bacteria. For example, the insertion sites of the one or more genes in the genome of the engineered bacteria satisfy the following:

[0065] (a) Located outside the transcriptional unit of a protein-coding gene;

[0066] (b) at least 50 kb from the transcription start site of any endogenous gene; and / or,

[0067] (c) Not located inside any known microRNA (miRNA) or long noncoding RNA (lncRNA) gene.

[0068] In some embodiments, the method includes exogenously introducing one or more copies of the genes MET13, DFR1, FOL3, and / or ERC1 into the *Bacillus thuringiensis*. Those skilled in the art will readily understand that it is not required that the individual copies of the genes have identical nucleotide sequences, as long as each copy can perform its original functional activity (e.g., achieve protein expression).

[0069] In some embodiments, the method includes exogenously introducing genes MET13, DFR1, FOL3 and / or ERC1 from Saccharomyces cerevisiae or Saccharomyces boulardii into the chassis bacteria.

[0070] In some embodiments, the method includes the exogenous introduction of genes MET13, DFR1, FOL3 and / or ERC1 from Saccharomyces cerevisiae (e.g., Saccharomyces cerevisiae S288C) into the chassis bacteria.

[0071] In some embodiments, the method includes the exogenous introduction of genes MET13, DFR1, FOL3 and / or ERC1 from Saccharomyces boulardii (Saccharomyces boulardii MYA-796) into the chassis bacteria.

[0072] In some embodiments, the substrate bacteria are yeasts. In some embodiments, the substrate bacteria are selected from: *Saccharomyces boulardii*, *Saccharomyces pastorianus*, *Pichiastipitis*, *Saccharomyces Bayanus*, and *Candidashehatae*. In some embodiments, the substrate bacteria are *Saccharomyces boulardii*, for example, *Saccharomyces boulardii* MYA-796.

[0073] In the fourth aspect, this application provides Saccharomyces boulardii Sb-M13, which was deposited on April 8, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number 34120.

[0074] In a fifth aspect, this application provides the use of the engineered strain of the first or second aspect, or Saccharomyces boulardii Sb-M13 of the fourth aspect, or the fermentation product of said engineered strain or Saccharomyces boulardii Sb-M13 in the preparation of 5-methyltetrahydrofolate or its salt.

[0075] In a sixth aspect, this application provides a method for preparing 5-methyltetrahydrofolate or a salt thereof, comprising:

[0076] (1) Cultivate engineered bacteria of the first or second aspect or Saccharomyces boulardii Sb-M13 of the fourth aspect;

[0077] (2) Obtain (e.g., by isolation and purification) 5-methyltetrahydrofolate or its salts from the culture of the engineered bacteria or Saccharomyces boulardii Sb-M13.

[0078] In a seventh aspect, this application provides the use of the engineered strain of the first or second aspect, or Saccharomyces boulardii Sb-M13 of the fourth aspect, or the fermentation product of said engineered strain or Saccharomyces boulardii Sb-M13, in the preparation of a product for supplementing 5-methyltetrahydrofolate or a salt thereof in a subject. In some embodiments, said product is a pharmaceutical, health product, or food.

[0079] In some implementations, the product is administered to the subject orally.

[0080] In some implementations, the product is a microbial agent.

[0081] In some embodiments, the microbial agent comprises the engineered bacteria in live cell form or the Saccharomyces boulardii Sb-M13.

[0082] In some embodiments, the microbial agent contains at least 1 × 10⁻⁶ 2 CFU / mL (e.g., at least 1×10⁻⁶) 3 CFU / mL, at least 1×10 4 CFU / mL, at least 1×10 5 CFU / mL, at least 1×10 6 CFU / mL, at least 1×10 7 CFU / mL, at least 1×10 8 CFU / mL, at least 1×10 9 The engineered bacteria (CFU / mL) or the Saccharomyces boulardii Sb-M13.

[0083] In some embodiments, the microbial agent further comprises strains selected from: Lactobacillus plantarum LP45, Lactobacillus reuteri L840, Bifidobacterium bifidum B11, Bifidobacterium longum subsp. longum L693, Lactobacillus rhamnosus LR519, and any combination thereof.

[0084] In some implementations, the subject is a mammal, such as a human.

[0085] In some implementations, the subject possesses characteristics such as MTHFR (methylenetetrahydrofolate reductase) gene mutation, old age, and / or digestive disorders.

[0086] On the other hand, this application provides a method for supplementing a subject with 5-methyltetrahydrofolate or a salt thereof, comprising administering to the subject an engineered strain of the first or second aspect, or Saccharomyces boulardii Sb-M13 of the fourth aspect, or the engineered strain or fermentation product of Saccharomyces boulardii Sb-M13.

[0087] In some embodiments, the engineered bacteria or Saccharomyces boulardii Sb-M13 or fermentation products are administered to the subject orally.

[0088] In some embodiments, the engineered bacteria or Saccharomyces boulardii Sb-M13 is administered to the subject in the form of an inoculum.

[0089] In some embodiments, the microbial agent comprises the engineered bacteria in live cell form or the Saccharomyces boulardii Sb-M13.

[0090] In some embodiments, the microbial agent contains at least 1 × 10⁻⁶ 2 CFU / mL (e.g., at least 1×10⁻⁶) 3 CFU / mL, at least 1×10 4 CFU / mL, at least 1×10 5 CFU / mL, at least 1×10 6 CFU / mL, at least 1×10 7 CFU / mL, at least 1×10 8 CFU / mL, at least 1×10 9 The engineered bacteria (CFU / mL) or the Saccharomyces boulardii Sb-M13.

[0091] In some embodiments, the microbial agent further comprises strains selected from: Lactobacillus plantarum LP45, Lactobacillus reuteri L840, Bifidobacterium bifidum B11, Bifidobacterium longum subsp. longum L693, Lactobacillus rhamnosus LR519, and any combination thereof.

[0092] In an eighth aspect, this application provides the use of the engineered strain of the first or second aspect, or Saccharomyces boulardii Sb-M13 of the fourth aspect, or the fermentation product of said engineered strain or Saccharomyces boulardii Sb-M13, in the preparation of a product for the prevention and / or treatment of Alzheimer's disease in subjects. In some embodiments, said product is a pharmaceutical, health product, or food.

[0093] In some implementations, the product is used for one or more of the following:

[0094] (i) Alleviating anxiety-like behaviors in subjects (e.g., Alzheimer's patients);

[0095] (ii) Enhance hippocampal-dependent memory consolidation processes in subjects (e.g., Alzheimer's patients);

[0096] (iii) Enhance the spatial memory retrieval and maintenance abilities of subjects (e.g., Alzheimer's patients);

[0097] (iv) Improve the prefrontal cortex-mediated working memory flexibility in subjects (e.g., Alzheimer's disease patients);

[0098] (v) Improve spatial learning ability in subjects (e.g., Alzheimer's patients);

[0099] (vi) To promote the long-term retention of spatial memory in subjects (e.g., Alzheimer's patients);

[0100] (vii) Improve cognitive impairment in subjects (e.g., patients with Alzheimer's disease).

[0101] In some implementations, the product is administered to the subject orally.

[0102] In some implementations, the product is a microbial agent.

[0103] In some embodiments, the microbial agent comprises the engineered bacteria in live cell form or the Saccharomyces boulardii Sb-M13.

[0104] In some embodiments, the microbial agent contains at least 1 × 10⁻⁶ 2 CFU / mL (e.g., at least 1×10⁻⁶) 3 CFU / mL, at least 1×10 4 CFU / mL, at least 1×10 5 CFU / mL, at least 1×10 6 CFU / mL, at least 1×10 7 CFU / mL, at least 1×10 8 CFU / mL, at least 1×10 9 The engineered bacteria (CFU / mL) or the Saccharomyces boulardii Sb-M13.

[0105] In some embodiments, the microbial agent further comprises strains selected from: Lactobacillus plantarum LP45, Lactobacillus reuteri L840, Bifidobacterium bifidum B11, Bifidobacterium longum subsp. longum L693, Lactobacillus rhamnosus LR519, and any combination thereof.

[0106] In some embodiments, the product is administered in combination with additional components (e.g., pharmaceutically active agents) that have activity in preventing and / or treating Alzheimer's disease.

[0107] In some implementations, the subject is a mammal, such as a human.

[0108] On the other hand, this application provides a method for preventing and / or treating Alzheimer's disease in a subject, comprising administering to the subject an engineered bacterium of the first or second aspect, or Saccharomyces boulardii Sb-M13 of the fourth aspect, or a fermentation product of the engineered bacterium or Saccharomyces boulardii Sb-M13.

[0109] In some implementations, the method is used for one or more of the following:

[0110] (i) Alleviating anxiety-like behaviors in subjects (e.g., Alzheimer's patients);

[0111] (ii) Enhance hippocampal-dependent memory consolidation processes in subjects (e.g., Alzheimer's patients);

[0112] (iii) Enhance the spatial memory retrieval and maintenance abilities of subjects (e.g., Alzheimer's patients);

[0113] (iv) Improve the prefrontal cortex-mediated working memory flexibility in subjects (e.g., Alzheimer's disease patients);

[0114] (v) Improve spatial learning ability in subjects (e.g., Alzheimer's patients);

[0115] (vi) To promote the long-term retention of spatial memory in subjects (e.g., Alzheimer's patients);

[0116] (vii) Improve cognitive impairment in subjects (e.g., patients with Alzheimer's disease).

[0117] In some embodiments, the engineered bacteria, or Saccharomyces boulardii Sb-M13, or fermentation product is administered to the subject orally.

[0118] In some embodiments, the engineered bacteria, or Saccharomyces boulardii Sb-M13, is administered to the subject in the form of an inoculum.

[0119] In some embodiments, the microbial agent comprises the engineered bacteria in live cell form or the Saccharomyces boulardii Sb-M13.

[0120] In some embodiments, the microbial agent contains at least 1 × 10⁻⁶ 2 CFU / mL (e.g., at least 1×10⁻⁶) 3 CFU / mL, at least 1×10 4 CFU / mL, at least 1×10 5 CFU / mL, at least 1×10 6 CFU / mL, at least 1×10 7 CFU / mL, at least 1×10 8 CFU / mL, at least 1×10 9 The engineered bacteria (CFU / mL) or the Saccharomyces boulardii Sb-M13.

[0121] In some embodiments, the microbial agent further comprises strains selected from: Lactobacillus plantarum LP45, Lactobacillus reuteri L840, Bifidobacterium bifidum B11, Bifidobacterium longum subsp. longum L693, Lactobacillus rhamnosus LR519, and any combination thereof.

[0122] In some embodiments, the engineered bacteria, Saccharomyces boulardii Sb-M13, or fermentation product are administered in combination with other components (e.g., pharmaceutically active agents) that have activity in preventing and / or treating Alzheimer's disease.

[0123] In some implementations, the subject is a mammal, such as a human.

[0124] In some implementations, the subject possesses characteristics such as MTHFR (methylenetetrahydrofolate reductase) gene mutation, old age, and / or digestive disorders.

[0125] In a ninth aspect, this application provides a microbial agent comprising the engineered bacteria or the Saccharomyces boulardii Sb-M13 in live cell form.

[0126] In some embodiments, the microbial agent contains at least 1 × 10⁻⁶ 2 CFU / mL (e.g., at least 1×10⁻⁶) 3 CFU / mL, at least 1×10 4 CFU / mL, at least 1×10 5 CFU / mL, at least 1×10 6 CFU / mL, at least 1×10 7 CFU / mL, at least 1×10 8 CFU / mL, at least 1×10 9 The engineered bacteria (CFU / mL) or the Saccharomyces boulardii Sb-M13.

[0127] In some embodiments, the microbial agent further comprises strains selected from: Lactobacillus plantarum LP45, Lactobacillus reuteri L840, Bifidobacterium bifidum B11, Bifidobacterium longum subsp. longum L693, Lactobacillus rhamnosus LR519, and any combination thereof.

[0128] In a tenth aspect, this application provides a pharmaceutical composition comprising the engineered bacteria of the first or second aspect, or Saccharomyces boulardii Sb-M13 of the fourth aspect, or the fermentation product of said engineered bacteria or Saccharomyces boulardii Sb-M13.

[0129] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0130] In some embodiments, the pharmaceutical composition further comprises additional components (e.g., pharmaceutically active agents) having activity in preventing and / or treating Alzheimer's disease.

[0131] In the eleventh aspect, this application provides the use of the engineered strain of the first or second aspect or the fourth aspect, *Saccharomyces boulardii* Sb-M13, in constructing engineered strains for the prevention and / or treatment of Alzheimer's disease or for the preparation of 5-methyltetrahydrofolate or its salts.

[0132] Terminology Definition

[0133] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virological, biochemical, and immunological laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0134] When the terms “for example,” “such as,” “like,” “including,” “contains,” or variations thereof are used herein, these terms will not be considered restrictive terms but will be interpreted as meaning “but not limited to” or “not limited to.”

[0135] Unless otherwise specified herein or clearly contradicted by the context, the terms “an” and “a kind” as well as “the” and similar designations shall be interpreted to cover both the singular and the plural in the context of describing the invention (especially in the context of the following claims).

[0136] As used herein, the term "engineered bacteria" refers to a bacterial strain that has undergone artificial modifications or alterations. Typically, the genome of such engineered bacteria typically includes artificially introduced modifications or alterations. These modifications or alterations include, but are not limited to, the insertion, deletion, substitution, or regulation of genes, designed to confer functional or phenotypic changes on the strain compared to its natural form.

[0137] As used in this article, the term "chassis bacteria" (or "chassis cells") refers to the microbial strains that serve as the starting point for genetic manipulation or metabolic engineering. These strains typically possess a well-defined genetic background and physiological characteristics, providing the necessary intracellular environment, cofactor supply, and gene expression regulation basis for the introduction, assembly, and optimization of heterologous or homologous metabolic pathways.

[0138] As used herein, the terms "5-methyltetrahydrofolate" and "5-MTHF" refer to the active form of folic acid, which is the compound N-[4-[[(2-amino-1,4,5,6,7,8-hexahydro-4-oxo-5-methyl-6-pteridyl)methyl]amino]benzoyl]-L-glutamic acid, preferably its naturally occurring, biologically active (6S)-stereoisomer. 5-Methyltetrahydrofolate is generally found in the form of salts, including salts formed with pharmaceutically or food-acceptable acids or bases, including but not limited to alkali metal salts, alkaline earth metal salts, and salts formed with amino acids or organic bases, such as methylcobalamin, calcium salts, etc.; and the term should also encompass various solvates, hydrates, and different crystalline forms of the compound or its salts.

[0139] As used herein, the term "prevention" refers to a method implemented to prevent or delay the onset of a disease, condition, or symptom (e.g., Alzheimer's disease) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include (but are not limited to) symptom relief, disease reduction, stabilization (i.e., no further deterioration) of the disease state, delay or slowing disease progression, improvement or relief of the disease state, and symptom relief (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to extended survival compared to expected survival (if no treatment was received).

[0140] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing a disease (e.g., Alzheimer's disease) means an amount sufficient to prevent, stop, or delay the onset of the disease (e.g., Alzheimer's disease); an effective amount for treating a disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art.

[0141] Beneficial effects of the invention

[0142] The engineered probiotics provided by this invention can stably, continuously, and efficiently produce 5-methyltetrahydrofolate, greatly reducing the production cost of 5-methyltetrahydrofolate, simplifying the process, and promoting the green and clean production of high-value pharmaceutical and biochemical products, which has important economic value and social significance.

[0143] Furthermore, the engineered probiotics provided by this invention can be significantly beneficial for the prevention and / or treatment of Alzheimer's disease, and experiments have confirmed that they can produce significant therapeutic effects in various mouse models of Alzheimer's disease.

[0144] Instructions on the Preservation of Biological Materials

[0145] This invention relates to the following biological materials that have been deposited at the China General Microbiological Culture Collection Center (CGMCC):

[0146] Saccharomyces boulardii Sb-M13, with accession number 34120 and accession date of April 8, 2025.

[0147] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0148] Figure 1 Image of YCplac33-Cas9 plasmid.

[0149] Figure 2 : pRS42H-gRNA plasmid map.

[0150] Figure 3 Results of the mining experiment: Left figure: Total distance traveled by mice in each group during the mining experiment; Right figure: Percentage of time spent in the central area by mice in each group during the mining experiment; The AD model mice used were 5FAD model mice.

[0151] Figure 4 Results of the mining experiment: Left figure: Total distance traveled by mice in each group during the mining experiment; Right figure: Percentage of time spent in the central area by mice in each group during the mining experiment; The AD model mice used were P301S model mice.

[0152] Figure 5 Results of the new object recognition experiment. The AD model mouse used in the left image is the 5FAD model mouse, and the AD model mouse used in the right image is the P301S model mouse.

[0153] Figure 6 Results of dwell time detection in the new and old arms of the Y-maze. Left figure: Percentage of dwell time in the new arm of the Y-maze; Right figure: Percentage of entry times into the new arm of the Y-maze; The AD model mouse used was the 5FAD model mouse.

[0154] Figure 7 Results of dwell time detection in the new and old arms of the Y-maze. Left figure: Percentage of dwell time in the new arm of the Y-maze; Right figure: Percentage of entry times into the new arm of the Y-maze; The AD model mouse used was the P301S model mouse.

[0155] Figure 8The results of the spontaneous alternation test in the Y-maze are shown on the left and right. The left figure shows the AD model mouse model of 5FAD mice, and the right figure shows the AD model mouse model of P301S mice.

[0156] Figure 9 : Results of Morris water maze behavioral testing. The left image shows the 5FAD model mouse; the right image shows the P301S model mouse.

[0157] Figure 10 Target quadrant exploration experiment. Left figure: Percentage of exploration distance in the target quadrant for each group of mice; Right figure: Percentage of exploration time in the target quadrant for each group of mice; The AD model mice used were 5FAD model mice.

[0158] Figure 11 Target quadrant exploration experiment. Left figure: Percentage of time spent exploring the target quadrant in each group of mice; Right figure: Percentage of distance spent exploring the target quadrant in each group of mice; The AD model mice used were P301S model mice. Detailed Implementation

[0159] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it). Those skilled in the art will appreciate that the embodiments are described by way of example and are not intended to limit the scope of protection claimed by the invention.

[0160] The following are some of the culture media involved in the embodiments of this application:

[0161] (1) LB (Luria Bertani)

[0162] Liquid: yeast extract (w / v): 0.5%, tryptic peptone (w / v): 1%, NaCl (w / v): 1%, adjust the pH of the culture medium to 7.5, and sterilize at 121℃ for 15 min.

[0163] Solid: Add agar powder to liquid LB medium to a final concentration of 1.5% (w / v), sterilize, and cool slightly before pouring into plates.

[0164] LBA medium: Add ampicillin to sterilized LB liquid or solid medium. The usual concentration of ampicillin is 100 μg / mL.

[0165] (2) YPD medium

[0166] Liquid: Yeast extract (w / v): 1%, peptone (w / v): 2%, sterilized at 121℃ for 15 min, then cooled to a temperature that is not hot to the touch, and 40% (w / v) glucose stock solution (to be sterilized separately: 115℃, 25 min) was added to make the final glucose concentration of the medium 2% (w / v).

[0167] Solid: Add agar powder to YPD liquid medium to a final concentration of 1.5% (w / v), sterilize and cool before pouring into plates.

[0168] (3) Complete Limit (CM) Component-Free Culture Medium

[0169] YNB (w / v): 0.67%, Dropout powder (w / v): 0.083%, and the final concentrations of its components are as follows (mg / L): threonine 150, serine 150, valine 150, glutamic acid 10, aspartic acid 100, phenylalanine 50, lysine 30, tyrosine 30, isoleucine 30, arginine 20, methionine 20;

[0170] Other nutrients (mg / L):

[0171] Histidine 100, Leucine 100, Tryptophan 100, Uracil 50, Adenine 50;

[0172] The pH range of liquid culture medium is 5.6 to 5.8; the pH range of solid culture medium is around 6.5.

[0173] (4) CMG culture medium

[0174] Liquid culture medium: CMG medium is made by adding glucose stock solution to CM medium to a final concentration of 2%.

[0175] Solid culture medium: Add agar powder (w / v) to liquid CMG medium at a final concentration of 1.5%.

[0176] CMG+HyB 300 solid medium: Add an appropriate amount of hygromycin B to CMG liquid or solid medium to prepare antibiotic screening plates.

[0177] In addition, by removing some amino acids from the CM medium, a auxotrophic screening medium / plate can be prepared.

[0178] (5) 5-Fluororhodic acid medium (5'-FOA)

[0179] Solution I: Prepare 50 mL of 5'-FOA solution, comprising: YNB AA (YNB with Amino Acids): 0.7 g, dropout powder: 0.085 g, 5'-FOA: 0.1 g, uracil: 10 mg, adenine: 5 mg, leucine: 20 mg, histidine: 15 mg, tryptophan: 10 mg, glucose: 2 g; dissolve thoroughly at 45 °C, filter sterilize, and store.

[0180] Solution II: Prepare 50 mL of agar powder solution with a final concentration of 3%, sterilize at 121°C for 15 min, and then cool to 45°C. Carefully mix Solution I and Solution II, avoiding the formation of air bubbles, and quickly pour into sterile petri dishes. Allow to cool and solidify before use.

[0181] The primer sequences involved in the embodiments of this application are shown in Table 1-1.

[0182] Table 1-1 Primer sequences used in the examples

[0183] The gene information involved in the embodiments of this application is shown in Table 1-2 below:

[0184] Table 1-2 Exemplary Gene Information

[0185] The gRNA target sites involved are shown in Table 1-3:

[0186] Table 1-3 gRNA and target information

[0187] Example 1: Construction of a yeast strain with the fol3 gene inserted

[0188] The fol3 gene was inserted into the yeast chromosome using the CRISPR / Cas9 system. First, the Cas9 protein vector plasmid YCplac33-Cas9 (carrying the selection marker URA3) was transformed into competent yeast cells (Saccharomyces boulardii MYA-796; referred to as SB in this paper) using the lithium acetate method. Positive yeast transformants carrying the Cas9 plasmid were then selected using URA3 as the selection marker. Next, pRS42H-gfol3, which guides Cas9 protein cleavage at a specific site, and a donor fragment for DNA strand repair were constructed. The gRNA expression plasmid and the donor fragment were then transformed into competent yeast cells. Positive transformants selected using URA3 and hygromycin as dual selection markers were verified by PCR, and the yeast strain SB-FOL3, which underwent homologous recombination, was identified.

[0189] The primers used for construction are shown in Table 1, and the schematic diagram is shown in [reference needed]. Figure 1-2 .

[0190] 1. Construction of pRS42H-gfol3 plasmid

[0191] This embodiment uses the linearized pRS42H-gRNA-NotI plasmid digested with NotI as a template. DNA polymerase from Novizan was used, and the PCR fragment was obtained by annealing at 54°C for 15 seconds and extending at 72°C for 4 minutes for 30 cycles. A 6600bp PCR fragment was then obtained. The PCR fragment was self-ligated using a seamless recombination kit, resulting in the ligation of homologous sequences at both ends to form the pRS42H-gRNA-ORF plasmid (ORF being the gene targeting the target site). 10V of the plasmid was transformed into competent E. coli cells. After positive transformants grew on selection plates (LB+AMP100), the transformant colonies were expanded in LBA liquid medium and incubated overnight at 37°C and 230 rpm. The plasmid was then extracted and sequenced for verification. The resulting plasmid was pRS42H-gfol3.

[0192] Part 2: Construction of FOL3 donor fragments

[0193] Using *Saccharomyces cerevisiae* chromosome as a template, the left homologous arm fragment of H1 was obtained by PCR using primer pairs P3 and P4 in the primer list; the TDH3 promoter fragment was obtained by PCR using primer pairs P5 and P6 in the primer list; the Fol3 gene fragment was obtained by PCR using primer pairs P7 and P8 in the primer list; the TEF1 terminator fragment was obtained by PCR using primer pairs P9 and P10 in the primer list; and the right homologous arm fragment of H2 was obtained by PCR using primer pairs P11 and P12 in the primer list.

[0194] The H1 left homologous arm fragment, TDH3 promoter fragment, Fol3 gene fragment, TEF1 terminator fragment, and H2 right homologous arm fragment were mixed in a certain proportion and subjected to PCR using DNA polymerase produced by Novizan. The reaction was carried out at 54°C for 15 seconds, with an extension time of 2 minutes, for 11 cycles. 1 μL of the reaction mixture was then used as the template for the next PCR reaction. Using P3 and P12 from Table 1-1 as upstream and downstream primers, the PCR reaction was carried out at 54°C for 15 seconds, with an extension time of 2 minutes, for 35 cycles. The PCR products were purified using a gel extraction kit to obtain the FOL3 donor fragment.

[0195] FOL3 donor fragment construction is referenced in Table 2-1.

[0196] Table 2-1

[0197] 3: Construction of SB-FOL3 strain

[0198] The YCplac33-Cas9 plasmid was transformed into *Saccharomyces boulardii* SB competent cells, and positive transformants were selected using the URA3 selection marker. After culturing the positive transformants into competent cells, the pRS42H-gFOL3 plasmid and the FOL3 donor fragment were added, and positive transformants were selected again using the URA3 and hygromycin selection markers. The positive transformants were then cultured in YPD liquid culture, and chromosomal DNA was extracted and used as a template for PCR verification. The PCR products were then sent for assay. Integration was successful, and the original 5 bp insertion site was replaced with the FOL3 target gene. The positive transformant strain was named SB-FOL3.

[0199] Example 2: Construction of a yeast strain with inserted DFR1 gene

[0200] 1. Construction of pRS42H-gDFR1 plasmid

[0201] The DFR1 gene was inserted using plasmid pRS42H-gDFR1. The construction process was similar to that of pRS42H-gFOL3 in Example 1, using primer pairs P13 and P14 from Table 1-1. The PCR product was a 6600bp DNA fragment. The resulting plasmid was pRS42H-gDFR1.

[0202] Part 2: Construction of DFR1 donor fragments

[0203] The construction of the DFR1 donor fragment for DFR1 gene insertion was similar to the construction of the FOL3 donor fragment in Example 1. The primer pair used for synthesizing the left homologous arm of H1 was P15 and P16 from Table 1-1; the primer pair used for synthesizing the TEF1 promoter was P17 and P18 from Table 1-1; the primer pair used for synthesizing the DFR1 gene was P19 and P20 from Table 1-1; the primer pair used for synthesizing the TDH3 terminator was P21 and P22 from Table 1-1; and the primer pair used for synthesizing the right homologous arm of H2 was P23 and P24 from Table 1-1. The primer pair used for synthesizing the DFR1 donor fragment was P15 and P24 from Table 1-1.

[0204] DFR1 donor fragment construction is referenced in Table 2-2.

[0205] Table 2-2

[0206] 3: Construction of SB-FOL3-DFR1 strain

[0207] Based on the SB-FOL3 strain successfully constructed in Example 1, the YCplac33-Cas9 plasmid was transformed into *Saccharomyces boulardii* SB-FOL3 competent cells. Positive transformants were selected using the URA3 selection marker. After culturing the positive transformants into competent cells, the pRS42H-gDFR1 plasmid and the DFR1 donor fragment were added. Positive transformants were again selected using the URA3 and hygromycin selection markers. The positive transformants were then cultured in YPD liquid culture, and chromosomal DNA was extracted and used as a template for PCR verification. The PCR products were then sequenced for verification. Integration was successful, and the 5 bp fragment at the original genome insertion site was replaced with a 2509 bp DFR1 target gene carrying an expression cassette. The positive transformant strain was named SB-FOL3-DFR1.

[0208] Example 3: Construction of a yeast strain overexpressing the ERC1 gene

[0209] 1. Construction of pRS42H-g ERC1 plasmid

[0210] The ERC1 gene was inserted using plasmid pRS42H-gERC1. The construction process was similar to that of pRS42H-gFOL3 in Example 1, using primer pairs P25 and P26 from Table 1-1. The PCR product was a 6600bp DNA fragment. The resulting plasmid was pRS42H-gERC1.

[0211] Part 2: Construction of ERC1 donor fragment

[0212] Construction of the ERC1 donor fragment for ERC1 gene insertion. The primer pair used for synthesizing the left homologous arm of H1 is P27 and P28 from Table 1-1; the primer pair used for synthesizing the PGK1 promoter is P29 and P30 from Table 1-1; the primer pair used for synthesizing the ERC1 gene is P31 and P32 from Table 1-1; the primer pair used for synthesizing the PGK1 terminator is P33 and P34 from Table 1-1; and the primer pair used for synthesizing the right homologous arm of H2 is P35 and P36 from Table 1-1. The primer pair used for synthesizing the ERC1 donor fragment is P27 and P36 from Table 1-1.

[0213] Refer to Table 2-3 for ERC1 donor fragment construction.

[0214] Table 2-3

[0215] 3: Construction of SB-FOL3-DFR1-ERC1 strain

[0216] Based on the SB-FOL3-DFR1 strain successfully constructed in Example 2, the YCplac33-Cas9 plasmid was transformed into *Saccharomyces boulardii* SB-FOL3-DFR1 competent cells. Positive transformants were selected using the URA3 selection marker. After culturing the positive transformants into competent cells, the pRS42H-g ERC1 plasmid and the ERC1 donor fragment were added. Positive transformants were again selected using the URA3 and hygromycin selection markers. The positive transformants were then cultured in YPD liquid culture, and chromosomal DNA was extracted and used as a template for PCR verification. The PCR products were then sequenced for verification. Integration was successful, and the 5 bp insertion site in the original genome was replaced with a 3390 bp ERC1 target gene carrying the expression cassette. The positive transformant strain was named SB-FOL3-DFR1-ERC1.

[0217] Example 4: Construction of a yeast strain overexpressing the MET13 gene

[0218] 1. Construction of pRS42H-gMET13 plasmid

[0219] The MET13 gene was inserted using plasmid pRS42H-gMET13. The construction process was similar to that of pRS42H-gFol3 in Example 1, using primer pairs P37 and P38 from Table 1-1. The PCR product was a 6600 bp DNA fragment. The resulting plasmid pRS42H-gMET13 was obtained.

[0220] II: MET13 Donor Fragment Construction

[0221] The construction of the MET13 donor fragment for MET13 gene insertion was similar to that of the Fol3 donor fragment in Example 1. The primer pairs used for synthesizing the left homologous arm of H1 were P39 and P40 (Table 1-1); the primer pairs used for synthesizing the TPI1 promoter were P41 and P42 (Table 1-1); the primer pairs used for synthesizing MET13 were P43 and P44 (Table 1-1); the primer pairs used for synthesizing the ADH1 terminator were P45 and P46 (Table 1-1); the primer pairs used for synthesizing the right homologous arm of H2 were P47 and P48 (Table 1-1); and the primer pairs used for synthesizing the donor fragment were P39 and P48 (Table 1-1).

[0222] MET13 donor fragment construction is referenced in Table 2-4.

[0223] Table 2-4

[0224] 3: Construction of SB-FOL3-DFR1-ERC1-MET13 strain

[0225] Based on the SB-FOL3-DFR1-ERC1 strain successfully constructed in Example 2, the YCplac33-Cas9 plasmid was transformed into Saccharomyces boulardii SB-FOL3-DFR1-ERC1 competent cells. Positive transformants were selected using the URA3 selection marker. After culturing the positive transformants into competent cells, the pRS42H-gMET13 plasmid and the MET13 donor fragment were added. Positive transformants were again selected using the URA3 and hygromycin selection markers. The positive transformants were then subjected to YPD liquid culture, and chromosomal DNA was extracted and used as a template for PCR verification. The PCR products were then sequenced for verification. Integration was successful, replacing the original 5 bp insertion site with a 3363 bp target gene carrying the MET13 expression cassette. The positive transformant strain was named SB-FOL3-DFR1-ERC1-MET13, which is Saccharomyces boulardii Sb-M13 deposited at CGMCC with accession number 34120.

[0226] Evaluation of the ability of four genetically modified yeast strains to produce 5-methyltetrahydrofolate

[0227] The fermentation production of 5-methyltetrahydrofolate by the above four yeast strains with different gene modifications was evaluated, and the operation was as follows:

[0228] 1. Seed culture: Pick colonies growing on YPD solid medium plates and inoculate them into test tubes containing 5 mL of YPD culture medium. Incubate overnight at 37°C and 220 rpm. If necessary, perform a second scale-up culture.

[0229] 2. Fermentation: Fermentation medium: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, natural pH; inoculate fresh seed culture into a 100 mL volumetric flask containing 25 mL of fermentation medium, control the initial OD600 value at around 0.1, and ferment at 37 °C and 220 rpm for 3 days.

[0230] 3. The sample needs to be centrifuged to collect bacterial cells, then thermally / ultrasonically disrupted, and extracted with cold buffer containing sodium ascorbate (pH 7.0–7.5) in the dark. The sample is then purified by anion exchange resin and concentrated by filtration.

[0231] 4. HPLC uses a C18 reversed-phase column with phosphate buffer-methanol / acetonitrile as the mobile phase. Detection is performed at 290 nm UV or 295 / 360 nm fluorescence wavelengths. Strict light protection and low temperature operation are required throughout the process, and sample injection must be completed within 30 minutes to prevent degradation.

[0232] The evaluation of 5-methyltetrahydrofolate production by fermentation for 144 h is shown in Table 3.

[0233] Table 3. Evaluation of 5-methyltetrahydrofolate production by four different genetically modified yeast strains and SB Chasmozyme.

[0234] Example 5: 5-Methyltetrahydrofolate-engineered bacteria improve cognitive impairment in AD mice

[0235] 1. Experiment Overview

[0236] This embodiment systematically evaluated the effect of 5-methyltetrahydrofolate-engineered bacteria intervention on cognitive function by constructing an Alzheimer's disease (AD) mouse model. Two transgenic mouse types, 5FAD (Cyagen Biosciences, C001920) and P301S (Cyagen Biosciences, C001836), were used in the experiment. The groups included an engineered bacteria treatment group (“M”, AD model mice + engineered bacteria SB-FOL3-DFR1-ERC1-MET13), a basal bacterial treatment group (“SB”, AD model mice + basal bacterial Saccharomyces boulardii), a model control group (“AD”, AD mouse model + PBS), and a wild-type control group (“WT”, healthy mouse model + PBS). Mice in the basal bacterial treatment group received 200 μL of 1×10⁻⁶ bacteria daily. 9 CFU / mL of basal bacteria. Mice in the engineered bacteria treatment group received 200 μL of 1×10⁻⁶ bacteria daily. 9 CFU / mL engineered bacteria. The model control group and wild-type control group received 200 μL PBS daily. Administration was via gavage. Ten mice were in each group.

[0237] After 12 weeks of continuous intervention, the following behavioral tests were conducted sequentially: (1) open field test (assessing anxiety and spontaneous activity); (2) new object recognition test (assessing recognition memory); (3) Y-maze spontaneous alternation and new and old arm test (assessing working memory and spatial exploration); (4) Morris water maze (assessing spatial learning and memory). The experimental methods are as follows:

[0238] (1) Open Field Test (OFT)

[0239] Objective: To assess spontaneous activity, exploratory behavior, and anxiety-like behavior.

[0240] Principle: AD animals often exhibit reduced activity or increased anxiety (such as reduced exploration of the central region).

[0241] Equipment: A square open box (mice: 40×40×30cm; rats: 100×100×40cm), with the bottom divided into a central area and a peripheral area, and a camera on the top for recording.

[0242] step:

[0243] Adaptation to the environment: Animals were allowed to adapt to the test room for 30 minutes.

[0244] Place the animal: Gently place the animal in the center of the open box.

[0245] Record behavior: Record for 5-10 minutes (5 minutes is commonly used).

[0246] Analysis indicators:

[0247] Total distance traveled (cm) → Spontaneous activity

[0248] Calculation method: The total path length (unit: cm) of the animal moving in the open field within a set time (e.g., 5 minutes) is automatically calculated using video tracking software (such as EthoVision).

[0249] Formula: Integrating and summing the coordinates of consecutive points around the animal's center point (such as the tip of the nose or the center of gravity): Total distance = Σ√[(x_{i+1} - x_i)^2 + (y_{i+1} - y_i)^2] (where i is the time point)

[0250] Percentage of time spent in the central area / distance traveled → Anxiety level (lower values ​​indicate higher anxiety)

[0251] Definition: The central area is usually defined as the central area of ​​the open field (such as the area after removing the surrounding 20% ​​boundary).

[0252] Calculation formula:

[0253] Central area time percentage = (Central area time / Total test time) × 100%

[0254] Central area distance percentage = (Central area distance / Total distance traveled) × 100%

[0255] (2) Novel Object Recognition (NOR) Experiment

[0256] Objective: To assess recognition memory (non-spatial memory).

[0257] Principle: Animals are naturally inclined to explore new objects, and animals with impaired memory cannot distinguish between new and old objects.

[0258] step:

[0259] Phase 1: Adaptation period (24 hours prior)

[0260] Animals are placed alone in an open area for 5-10 minutes to familiarize themselves with the environment.

[0261] Phase 2: Training Phase (Session 1)

[0262] Place two identical objects (A1, A2) and allow them to explore freely for 5-10 minutes.

[0263] Record the total exploration time (nose is less than 2cm away from the object or touches it).

[0264] Phase 3: Testing Phase (Session 2)

[0265] Short-term memory (1-2 hours later): Replace one of the objects with a new object B (A vs B).

[0266] Long-term memory (after 24 hours): Same as above.

[0267] Animals are allowed to explore freely for 5 minutes.

[0268] Analysis indicators:

[0269] Recognition Index (RI)

[0270] Definition: The proportion of time an animal spends exploring new objects to the total exploration time (new + old objects).

[0271] formula:

[0272] RI = [T_novel / (T_novel + T_familiar)] × 100%

[0273] T_novel: Total time (seconds) to explore new objects.

[0274] T_familiar: Total time (seconds) spent exploring familiar objects.

[0275] (3) Y Maze (Spontaneous Alternating Task)

[0276] Objective: To assess working memory and spatial exploration abilities.

[0277] Principle: Healthy animals tend to explore new arms (with a high rate of spontaneous alternation), while animals with impaired working memory repeatedly enter the same arm.

[0278] step:

[0279] Maze structure: Three arms of equal length (angle 120°), arm length 30-40cm (mouse), arm width 5-10cm.

[0280] Adaptation: Animals can freely explore the maze for 5 minutes (optional).

[0281] Formal test:

[0282] Place the animals in the center of the maze.

[0283] Allow 8 minutes of free exploration and record the order of entering the arm (entering the arm completely counts as one time).

[0284] Analysis indicators:

[0285] Spontaneous Alternation Rate (%)

[0286] Definition: The proportion of animals entering three different arms in succession (e.g., in the order of A→B→C or A→C→B).

[0287] Calculation method:

[0288] Record the order in which the animal enters the arm (e.g., A, B, C, A, B, A → valid alternation sequence: ABC, BCA, CAB).

[0289] Valid alternation count: The number of times you enter different arms three times in a row (e.g., A→B→C counts as 1 time).

[0290] formula:

[0291] Spontaneous alternation rate = [Number of effective alternations / (Total number of arm entries - 2)] × 100%

[0292] The denominator is the total number of possible alternations (e.g., if there are 10 total entries, then the number of opportunities is 8).

[0293] Total Arm Entries

[0294] Definition: The total number of times the animal enters the arm during the test (the term "enter" must be clearly defined as: the whole body entering the arm).

[0295] (4) Novel Arm Y-Maze Test

[0296] Objective: To assess working memory and spatial exploration (more proactive than spontaneous alternation).

[0297] Principle: Animals prefer to explore new open arms, relying on their memory of previous explorations.

[0298] step:

[0299] Phase 1 (Familiarization Period):

[0300] Lock down one arm of the Y maze (let's call it arm C).

[0301] Animals were allowed to freely explore the two open arms (A and B) for 5 minutes.

[0302] Phase Two (Testing Period, with intervals of 30 minutes to 24 hours):

[0303] Open all three arms (A, B, C), where C is the new foreign body arm.

[0304] Animals are allowed to explore freely for 5 minutes.

[0305] Analysis indicators:

[0306] New Arm Time Ratio

[0307] formula:

[0308] New arm time percentage = (T_novel / T_total) × 100%

[0309] T_novel: Duration of stay on the new arm (seconds)

[0310] T_total: Total dwell time (seconds) across the three arms.

[0311] Novel Arm Entry Ratio

[0312] formula:

[0313] Percentage of new arm entry entries = (E_novel / E_total) × 100%

[0314] E_novel: Number of times entering the new arm

[0315] E_total: Total number of times entered all arms

[0316] (5) Morris Water Maze (MWM)

[0317] Objective: To assess spatial learning and reference memory (hippocampal dependence).

[0318] Equipment: Circular pool (1-2m in diameter), water temperature 22±1℃, milky white water (with added milk powder or titanium dioxide), hidden platform (1-2cm below the water surface), top tracking system.

[0319] step:

[0320] Phase 1: Visible platform training (optional, 1 day)

[0321] The platform emerges from the water, covered by a prominent logo.

[0322] Train the animal 4-5 times to ensure it understands the task (escape from the water).

[0323] Phase 2: Covert Platform Training (Spatial Learning, 5-7 days)

[0324] The platform is hidden in a fixed quadrant (such as the NE quadrant).

[0325] Train 4 times a day, starting from different entry points (N / S / E / W), with each session lasting a maximum of 60 seconds.

[0326] Once an animal finds the platform, it stays there for 15 seconds; if it does not find the platform, it is guided there.

[0327] Records: Escape incubation period (time to find the platform), swimming route.

[0328] Escape Latency

[0329] Definition: The time (in seconds) required from the moment the animal enters the water until it reaches the hidden platform.

[0330] Calculation: The average of 4 trials per day during the training period (reflecting the learning curve).

[0331] Phase 3: Probe Trial (Days 5 / 7 / 8)

[0332] Remove the platform and place the animal into the water from the opposite side (e.g., point SW).

[0333] Record within 60 seconds:

[0334] Time spent / distance in the target quadrant

[0335] Number of times the original platform location was crossed

[0336] Swimming speed (excluding the influence of movement obstacles)

[0337] Testing indicators:

[0338] Probe Trial Specifications:

[0339] Percentage of time spent in the target quadrant (Target Quadrant Time)

[0340] = (Target quadrant dwell time / 60 seconds) × 100%

[0341] Platform Crossings

[0342] Definition: The number of times an animal's swimming trajectory crosses the original platform position (the platform area is usually a circular area with a diameter of 20cm).

[0343] Swim Speed: Swim Speed ​​= Total Swimming Distance / Swimming Time (cm / second)

[0344] 2. Results of the behavioral experiment

[0345] 2.1 Open Field Experiment

[0346] In the mine experiment, the distance traveled directly reflects the overall motor skill level and exploration willingness of mice in an unfamiliar, open environment. It measures the basic activity ability of mice in the absence of direct external stimulation. The experimental results are as follows: Figure 3-4 As shown, compared with the AD control group mice, the WT control group mice moved a longer distance, indicating that the WT group mice had stronger exploration drive and curiosity, and were willing to actively explore new environments. Mice in the 5-methyltetrahydrofolate engineered bacteria treatment group showed exploration drive and curiosity similar to those in the WT group mice. Compared with the WT control group, the AD model group mice spent significantly less time in the central region, indicating that the AD group animals exhibited clear anxiety-like behaviors rather than motor function deficits. Mice in the 5-methyltetrahydrofolate engineered bacteria treatment group spent a longer time in the central region than the model group, approaching wild-type levels, while their peripheral movement distance decreased. These results indicate that engineered bacteria intervention effectively alleviates AD-related anxiety-like behaviors and promotes proactive environmental exploration.

[0347] 2.2 New Object Recognition

[0348] The results are as follows Figure 5 As shown, the recognition index of the AD model group mice was significantly lower than that of the wild-type group, confirming that their novel object recognition memory was severely impaired. The recognition index of the engineered bacteria treatment group was improved, showing a difference from the model group, indicating that the 5-methyltetrahydrofolate engineered bacteria can partially reverse AD-related object recognition memory impairment by enhancing the hippocampus-dependent memory consolidation process.

[0349] 2.3 Y-maze test

[0350] 2.3.1 New and old arm stay

[0351] The results are as follows Figure 6-7 As shown, the AD group had a significantly lower proportion of new arm exploration time and number of attempts compared to the WT group, reflecting spatial memory retrieval impairment. The 5-methyltetrahydrofolate engineered bacteria treatment group showed recovery of new arm exploration time and number of attempts, showing a difference from the AD group, demonstrating that engineered bacteria intervention enhanced the ability to retrieve and maintain spatial memory.

[0352] 2.3.2 Spontaneous Alternation

[0353] The results are as follows Figure 8As shown, the spontaneous alternation rate in the AD group was significantly lower than that in the WT group, indicating working memory deficit; the alternation rate in the 5-methyltetrahydrofolate engineered bacteria treatment group was higher than that in the model group, suggesting that the engineered bacteria intervention improved the prefrontal cortex-mediated working memory flexibility.

[0354] 2.4 Morris Water Maze

[0355] 2.4.1 Incubation period of the escape platform

[0356] The results are as follows Figure 9 As shown, during the 5-day training period, the average escape latency in the AD group was consistently higher than that in the WT control group, indicating impaired spatial learning ability. The latency in the 5-methyltetrahydrofolate engineered bacteria treatment group was shorter than that in the model group and the chassis treatment group, demonstrating that engineered bacteria intervention significantly accelerated the spatial learning process.

[0357] 2.4.2 Exploration of the Target Quadrant

[0358] The results are as follows Figure 10-11 As shown, in the space exploration experiment after platform removal, the AD group had significantly less time and distance spent in the target quadrant than the wild type, indicating severe impairment of long-term spatial memory; the 5-methyltetrahydrofolate engineered bacteria treatment group showed improved time and distance spent in the target quadrant compared to the AD model group, proving that 5-methyltetrahydrofolate engineered bacteria treatment effectively promotes the long-term maintenance of spatial memory.

[0359] 3. Experimental Conclusions

[0360] The series of behavioral experiments in this embodiment consistently demonstrate that intervention with 5-methyltetrahydrofolate-engineered bacteria can significantly improve multidimensional cognitive functions such as anxiety-like behavior, working memory, object recognition memory, and spatial learning memory in AD model mice, thus verifying the feasibility and effectiveness of this engineered bacteria in treating Alzheimer's disease from a behavioral phenotypic perspective.

[0361] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. Engineered bacteria, which contain one or more gene modifications selected from the following: (a) Gene modifications that enhance the expression of the FOL3 gene; (b) Gene modifications that enhance the expression of the DFR1 gene; (c) Gene modifications that enhance the expression of the ERC1 gene; (d) Gene modifications that enhance the expression of the MET13 gene.

2. The engineered bacteria of claim 1, comprising: (1) The modification (a); (2) The modifications (a) and (b); (3) The modifications (a), (b) and (c); or, (4) Modifications (a), (b), (c) and (d).

3. The engineered bacteria of claim 1 or 2, wherein, The gene modifications that enhance gene expression described in (a) through (d) are each independently selected from: promoters that enhance gene expression, gene linkage or introduction into enhancers, gene copy number increase, and any combination thereof; Preferably, the gene modification that enhances gene expression described in (a) to (d) is to increase the copy number of the gene.

4. Engineered bacteria containing one or more exogenously introduced genes, said genes being selected from: MET13, DFR1, FOL3, ERC1, and any combination thereof.

5. The engineered bacteria of claim 4, comprising exogenously introduced: (i) Gene FOL3; (ii) Genes FOL3 and DFR1; (iii) Genes DFR1, FOL3, and ERC1; or, (iv) Genes MET13, DFR1, FOL3 and ERC1.

6. The engineered bacteria of claim 4 or 5, wherein, (a) The exogenously introduced genes MET13, DFR1, FOL3 and / or ERC1 each have one or more copies independently; and / or, (b) the exogenously introduced genes MET13, DFR1, FOL3 and / or ERC1 are derived from Saccharomyces cerevisiae or Saccharomyces boulardii.

7. The engineered bacteria according to any one of claims 1-6, wherein, The engineered bacteria is yeast; Preferably, the engineered bacteria are selected from: Saccharomyces boulardii, Saccharomyces pastorianus, Pichiastipitis, Saccharomyces Bayanus, and Candida shehatae. Preferably, the engineered bacteria is *Saccharomyces boulardii*.

8. A method for constructing the engineered bacteria according to any one of claims 1-7, comprising: (1) Provide chassis bacteria; and, (2) Introducing the Chameleon bacteria with the gene modification defined in any one of claims 1-3, or introducing one or more genes exogenously into the Chameleon bacteria, wherein the genes are selected from: MET13, DFR1, FOL3, ERC1, and any combination thereof; Preferably, step (2) includes: introducing one or more genes exogenously into the chassis bacteria, wherein the genes are selected from: MET13, DFR1, FOL3, ERC1, and any combination thereof; Preferably, step (2) includes the exogenous introduction of the chassis bacteria: (i) Gene FOL3; (ii) Genes FOL3 and DFR1; (iii) Genes DFR1, FOL3, and ERC1; or, (iv) Genes MET13, DFR1, FOL3, and ERC1; Preferably, the method includes introducing one or more copies of the gene MET13, DFR1, FOL3 and / or ERC1 exogenously into the spores; preferably, the method includes introducing the gene MET13, DFR1, FOL3 and / or ERC1 derived from Saccharomyces cerevisiae or Saccharomyces boulardii exogenously into the spores. Preferably, the substrate bacteria are yeasts; preferably, the substrate bacteria are selected from: Saccharomyces boulardii, Saccharomyces pastorianus, Pichiastipitis, Saccharomyces Bayanus, and Candida shehatae; preferably, the substrate bacteria are Saccharomyces boulardii.

9. Saccharomyces boulardii Sb-M13, which was deposited on April 8, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number 34120.

10. Use of the engineered strain of any one of claims 1-7, or the Saccharomyces boulardii Sb-M13 of claim 9, or the fermentation product of said engineered strain or Saccharomyces boulardii Sb-M13 in the preparation of 5-methyltetrahydrofolate or its salt.

11. A method for preparing 5-methyltetrahydrofolate or a salt thereof, comprising: (1) Cultivate the engineered strain of any one of claims 1-7 or the Saccharomyces boulardii Sb-M13 of claim 9; (2) Obtain (e.g., by isolation and purification) 5-methyltetrahydrofolate or its salts from the culture of the engineered bacteria or Saccharomyces boulardii Sb-M13.

12. Use of the engineered strain of any one of claims 1-7, or the Saccharomyces boulardii Sb-M13 of claim 9, or the fermentation product of said engineered strain or Saccharomyces boulardii Sb-M13 in the preparation of a product for supplementing 5-methyltetrahydrofolate or its salt in subjects; preferably, said product is a pharmaceutical, health product or food.

13. The use of claim 12, wherein, The product is administered to subjects orally.

14. The use of claim 12 or 13, wherein, The product is a microbial agent; Preferably, the bacterial agent comprises the engineered bacteria in live cell form or the Saccharomyces boulardii Sb-M13; Preferably, the microbial agent contains at least 1×10 2 CFU / mL (e.g., at least 1×10⁻⁶) 3 CFU / mL, at least 1×10 4 CFU / mL, at least 1×10 5 CFU / mL, at least 1×10 6 CFU / mL, at least 1×10 7 CFU / mL, at least 1×10 8 CFU / mL, at least 1×10 9 The engineered bacteria or the Saccharomyces boulardii Sb-M13 (CFU / mL); Preferably, the microbial agent further comprises strains selected from the following: Lactobacillus plantarum LP45, Lactobacillus reuteri L840, Bifidobacterium bifidum B11, Bifidobacterium longum subsp. longum L693, Lactobacillus rhamnosus LR519, and any combination thereof.

15. Use of the engineered strain of any one of claims 1-7, or the Saccharomyces boulardii Sb-M13 of claim 9, or the fermentation product of said engineered strain or Saccharomyces boulardii Sb-M13 in the preparation of a product for the prevention and / or treatment of Alzheimer's disease in subjects; preferably, said product is a pharmaceutical, health product, or food.

16. The use of claim 15, wherein, The product is administered to subjects orally.

17. The use of claim 15 or 16, wherein, The product is a microbial agent; Preferably, the bacterial agent comprises the engineered bacteria in live cell form or the Saccharomyces boulardii Sb-M13; Preferably, the microbial agent contains at least 1×10 2 CFU / mL (e.g., at least 1×10⁻⁶) 3 CFU / mL, at least 1×10 4 CFU / mL, at least 1×10 5 CFU / mL, at least 1×10 6 CFU / mL, at least 1×10 7 CFU / mL, at least 1×10 8 CFU / mL, at least 1×10 9 The engineered bacteria or the Saccharomyces boulardii Sb-M13 (CFU / mL); Preferably, the microbial agent further comprises strains selected from the following: Lactobacillus plantarum LP45, Lactobacillus reuteri L840, Bifidobacterium bifidum B11, Bifidobacterium longum subsp. longum L693, Lactobacillus rhamnosus LR519, and any combination thereof.

18. The use according to any one of claims 15-17, wherein, The product is administered in combination with other components (e.g., pharmaceutically active agents) that have activity in preventing and / or treating Alzheimer's disease.

19. An inoculum comprising the engineered bacteria or the Saccharomyces boulardii Sb-M13 in live cell form; Preferably, the microbial agent contains at least 1×10 2 CFU / mL (e.g., at least 1×10⁻⁶) 3 CFU / mL, at least 1×10 4 CFU / mL, at least 1×10 5 CFU / mL, at least 1×10 6 CFU / mL, at least 1×10 7 CFU / mL, at least 1×10 8 CFU / mL, at least 1×10 9 The engineered bacteria or the Saccharomyces boulardii Sb-M13 (CFU / mL); Preferably, the microbial agent further comprises strains selected from the following: Lactobacillus plantarum LP45, Lactobacillus reuteri L840, Bifidobacterium bifidum B11, Bifidobacterium longum subsp. longum L693, Lactobacillus rhamnosus LR519, and any combination thereof.

20. A pharmaceutical composition comprising the engineered strain of any one of claims 1-7, or the Saccharomyces boulardii Sb-M13 of claim 9, or the engineered strain or the fermentation product of Saccharomyces boulardii Sb-M13; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises additional components (e.g., pharmaceutically active agents) having activity in preventing and / or treating Alzheimer's disease.

21. Use of the engineered strain of any one of claims 1-7 or the Saccharomyces boulardii Sb-M13 of claim 9 in the construction of engineered strains for the prevention and / or treatment of Alzheimer's disease or for the preparation of 5-methyltetrahydrofolate or its salts.

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