Engineering probiotics and their use in the preparation of a medicament for preventing and treating depressive disorders

Abstract

The present invention relates to the field of microbiology and biotechnology, in particular, the present invention relates to an engineered bacterium, cultures, dietary supplements and pharmaceutical compositions comprising the same, and their use in the manufacture of a medicament. The present invention also provides methods of culturing the engineered bacterium and methods of producing equol.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and biotechnology, and more specifically, to an engineered bacterium, cultures comprising the bacterium, dietary supplements and pharmaceutical compositions thereof, and their use in the preparation of pharmaceuticals. The invention also provides methods for culturing the engineered bacterium and methods for producing equadol. Background Technology

[0002] Depressive disorder (DD) is one of the most common and burdensome mental disorders worldwide, and a leading cause of "disability life years (YLD)". Its core clinical manifestations include persistent low mood, loss of interest / anhéphane, often accompanied by changes in sleep, appetite, and psychomotor function. A significant proportion of patients also experience cognitive impairments such as attention and executive function, affecting their learning and work abilities.

[0003] The pathophysiology of depression has traditionally been guided by the "monoamine hypothesis." Traditional antidepressants, including tricyclic antidepressants (TCAs), monoamine oxidase inhibitors (MAOIs), selective serotonin reuptake inhibitors (SSRIs), and serotonin-norepinephrine reuptake inhibitors (SNRIs), generally operate within this theoretical framework, exerting their effects by enhancing the availability of 5-HT, NE, and DA at synapses. While they are generally superior to placebos in the acute phase, improve symptoms, and are used for relapse prevention, their efficacy is often moderate, with limitations such as delayed onset, limited remission rates, common residual symptoms, and a high risk of relapse. Research over the past decade has indicated that the pathogenesis of depression is multifaceted, including neuroinflammation, stress-hypothalamic-pituitary-adrenal (HPA) axis abnormalities, synaptic plasticity / neurogenesis insufficiency, and complex interactions between neurotransmitter systems (monoamines, glutamate, dopamine, etc.). Therefore, drugs targeting a single monoamine pathway are insufficient to cover the entire patient population with highly heterogeneous depression, which has driven the development of new drugs targeting neuroinflammation and the glutamate pathway.

[0004] In recent years, the "Gut-Brain Axis (GBA)" theory has provided a new perspective for the pathophysiological study of depression. Research shows that the gut microbiota can influence central nervous system activity through pathways such as the vagus nerve, immune factors, cytokines, short-chain fatty acids, and tryptophan metabolites, thereby regulating mood, cognition, and stress responses. Conversely, mental stress can also affect gut microbiota homeostasis through the hypothalamus-pituitary-adrenal axis and the autonomic nervous system, forming a complex network of interaction among gut microbiota, immunity, and neuroendocrine systems. Numerous clinical and animal studies have confirmed that gut microbiota imbalance (Dysbiosis) is common in patients with depression, characterized by a decrease in the proportion of beneficial bacteria such as Bacteroides and Lactobacillus, while a relative increase in inflammatory bacteria. This imbalance can induce increased intestinal mucosal barrier permeability, promote systemic inflammatory responses and central penetration of peripheral immune factors, thereby exacerbating neuroinflammation and mood disorders.

[0005] Within the gut-brain axis framework, gut microbial metabolites are considered important mediators of central nervous system function regulation. Among them, equol, a metabolite of soy isoflavones, has attracted widespread attention as a natural polyphenol compound with estrogen-like activity due to its unique biological activity. Equol is produced by specific bacteria in the gut through the reduction of daidzein, the main component of soy isoflavones; however, only about 30%–50% of individuals in the population possess an "equol producer" gut microbiota. This individual difference leads to significant variations in the endogenous levels of equol under natural conditions, thus limiting its potential neuroprotective and antidepressant applications. Numerous studies have shown that equol can mediate mood regulation in both the central and peripheral systems through multiple pathways. On the one hand, its molecular structure is similar to that of 17β-estradiol, which can selectively activate estrogen receptor β (ERβ), thereby affecting neuronal plasticity, synapsis and the expression of neurotrophic factors (such as BDNF); on the other hand, equol has significant anti-inflammatory and antioxidant effects, which can inhibit microglia activation and the release of pro-inflammatory factors (IL-6, TNF-α), and reduce the damage of neuroinflammation to the mood regulation circuit.

[0006] To date, there are no reports of other studies or applications of estrol-producing engineered probiotics in the prevention and treatment of depressive disorders. Summary of the Invention

[0007] The applicant of this invention, through extensive experimentation, has provided an engineered bacterium (HLE0252). Compared to bacteria modified in other ways, this engineered bacterium exhibits significantly increased equadol production and extremely high biocompatibility. Furthermore, the engineered bacterium of this invention demonstrates outstanding efficacy in improving mood disorders, effectively alleviating depression and anxiety in subjects.

[0008] Therefore, in a first aspect, the present invention provides an engineered bacterium that differs from its wild-type originating microorganism at least in that: (1) Overexpress the following gene: daidzein reductase gene dznr dihydrodaidzein racemase gene ddrc dihydrodaidzein reductase gene dhdr Tetrahydrodaidzein reductase gene thdr glucose dehydrogenase gene gdh and the beta-glucoside permease gene bglF ; (2) Low expression or no expression: the endogenous plasmid of the wild-type microorganism.

[0009] In some implementations, the bacteria are probiotics.

[0010] In some implementations, the bacteria is Escherichia coli.

[0011] In some implementations, the engineered bacteria eliminate or remove the endogenous plasmids of the wild-type microorganism.

[0012] In some implementations, low or no expression of the plasmid is achieved by knocking out, deleting, or knocking down the endogenous plasmid of the wild-type microorganism.

[0013] It's understandable that Escherichia coli (E. coli) Escherichia coli "E. coli" and "coliform bacteria" are two Chinese translations of the same bacterial species. They have the same meaning and can be used interchangeably.

[0014] Escherichia coli Nissle 1917 ( Escherichia coli Nissle 1917 (EcN) is a probiotic isolated from the feces of healthy individuals by Alfred Nissle in 1917. EcN is widely used clinically. In the pre-antibiotic era, it was used as an anti-infective drug to treat infectious diarrhea and its prognosis. Later, it was also used to treat non-infectious gastrointestinal disorders, such as chronic constipation or inflammatory bowel disease. EcN possesses many excellent properties, exhibiting high host safety and lacking pathogenic factors such as enterotoxins, hemolytic toxins, and cytotoxins. EcN itself carries two cryptic plasmids, pMUT1 and pMUT2.

[0015] In some embodiments, the wild-type microorganism is Escherichia coli Nissle 1917 (EcN).

[0016] In some embodiments, the engineered bacteria of the present invention are derived from EcN as the chassis bacteria, inheriting the probiotic characteristics of EcN, with a clear source, safe and reliable, and possessing good intestinal colonization and programmable metabolic capabilities, making it easy to load and accurately express therapeutic functions.

[0017] In some embodiments, the endogenous plasmids of the wild-type microorganism include pMUT1 and / or pMUT2.

[0018] In some embodiments, the engineered bacteria do not express the endogenous plasmids pMUT1 and pMUT2 of the EcN.

[0019] It is understood that the overexpressed genes described above in this invention can originate from any organism (e.g., plants, animals, or microorganisms of a different species from the wild-type microorganism), and are not limited to the specific sources of the embodiments; those skilled in the art can also choose any method in the prior art to achieve low or no expression of the gene. For example, through gene editing technology.

[0020] In some implementations, gene overexpression is either the overexpression of an endogenous gene or the heterologous expression of an exogenous gene.

[0021] In some embodiments, the engineered bacteria contain exogenous components compared to their derived wild-type microorganisms. dznr , ddrc , dhdr , thdr and / or gdh Heterogeneous expression.

[0022] In some embodiments, the engineered bacteria, compared to their derived wild-type microorganisms, contain... bglF Endogenous overexpression.

[0023] In some embodiments, the engineered bacteria comprise Snakella mucinosa, a bacterium derived from *Synapsia ulmoides*. Slackia isoflavoniconvertens )of dznr Heterogeneous expression.

[0024] In some embodiments, the engineered bacteria comprise Snakella mucinosa, a bacterium derived from *Synapsia ulmoides*. Slackia isoflavoniconvertens )of ddrc Heterogeneous expression.

[0025] In some embodiments, the engineered bacteria comprise Snakella mucinosa, a bacterium derived from *Synapsia ulmoides*. Slackia isoflavoniconvertens )of dhdrHeterogeneous expression.

[0026] In some embodiments, the engineered bacteria comprise Snakella mucinosa, a bacterium derived from *Synapsia ulmoides*. Slackia isoflavoniconvertens )of thdr Heterogeneous expression.

[0027] In some embodiments, the engineered bacteria comprise bacteria derived from *Priscilla megaterium* (…). Priestia megaterium )of gdh Heterogeneous expression.

[0028] In some implementations, the engineered bacteria are driven by a strong promoter. bglF Endogenous overexpression.

[0029] The term "strong promoter" refers to a promoter that has high affinity for RNA polymerase and can direct the synthesis of large amounts of mRNA, thus functioning by efficiently initiating transcription. Common types include lacP, trpP, tacP, λPL, CaMV promoters, and adenovirus late gene promoters. It is understood that various types of strong promoters exist in the art, and those skilled in the art can use any strong promoter sequence. In some embodiments, the strong promoter comprises or has a nucleotide sequence as shown in SEQ ID NO: 35.

[0030] In some implementations, the dznr Includes or has a nucleotide sequence as shown in SEQ ID NO: 28, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with it.

[0031] In some implementations, the ddrc Includes or has a nucleotide sequence as shown in SEQ ID NO: 29, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with it.

[0032] In some implementations, the dhdrIncludes or has a nucleotide sequence as shown in SEQ ID NO: 30, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with it.

[0033] In some implementations, the thdr Includes or has a nucleotide sequence as shown in SEQ ID NO: 31, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with it.

[0034] In some implementations, the gdh Includes or has a nucleotide sequence as shown in SEQ ID NO: 32, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with it.

[0035] In some implementations, the bglF Includes or has a nucleotide sequence as shown in SEQ ID NO: 33, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with it.

[0036] In some implementations, the engineered bacteria is Escherichia coli.

[0037] In some embodiments, the engineered bacteria are deposited at the China General Microbiological Culture Collection Center (CGMCC) and have accession number CGMCC No. 34947.

[0038] In another aspect, the present invention provides a method for constructing engineered bacteria as described above, the method comprising: Overexpression in EcN strain dznr, ddrc, dhdr, thdr and gdh Gene; Endogenous plasmids pMUT1 and pMUT2 are expressed at low levels or not at all in the EcN strain.

[0039] In some embodiments, gene overexpression is achieved by transfecting the overexpressed gene into the EcN strain via a vector.

[0040] In some implementations, low or no expression of the plasmid is achieved by knocking out, deleting, or knocking down the endogenous plasmid of the EcN strain.

[0041] In some implementations, the method includes: Transfection expression in EcN strain dznr, ddrc, dhdr, thdr and gdh The carrier of genes; Knockout, deletion, or knockdown of endogenous plasmids pMUT1 and pMUT2 in EcN strains. In some implementations, dznr、 and gdh The gene is expressed using the pETDuet-1 vector. In some implementations, expression... dznr、 and gdh The vector of the gene contains or has a nucleotide sequence as shown in SEQ ID NO: 2, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with it.

[0042] In some implementations, ddrc, dhdr and thdr The gene is expressed using the vector pCDFDuet. In some implementations, expression... ddrc, dhdr and thdr The vector of the gene contains or has a nucleotide sequence as shown in SEQ ID NO: 3, or has a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to it.

[0043] In some implementations, CRISPR / Cas9 is used to edit plasmids (knockout, deletion, or knockdown of EcN strain endogenous plasmids pMUT1 and pMUT2).

[0044] In another aspect, the present invention provides a culture comprising engineered bacteria as described above.

[0045] In some embodiments, the culture contains at least 50 mg / L, at least 80 mg / L, at least 120 mg / L, at least 200 mg / L, at least 300 mg / L, at least 400 mg / L, or at least 500 mg / L equadol.

[0046] In another aspect, the present invention provides a dietary supplement comprising engineered bacteria or cultures as described above.

[0047] In some embodiments, the dietary supplement is formulated for oral administration.

[0048] In some implementations, the dietary supplement also contains prebiotics.

[0049] In some embodiments, the dietary supplement may further comprise melatonin, omega-3 dietary supplements, or any combination thereof.

[0050] In some implementations, the dietary supplement also contains vitamins or minerals.

[0051] In some embodiments, the vitamins are selected from vitamin A (as all-trans retinol, all-trans retinyl ester, and all-trans β-carotene and other provitamin A carotenoids), vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B7 (biotin), vitamin B9 (folic acid or folic acid ester), vitamin B12 (cobalamin), vitamin C (ascorbic acid), vitamin D (calciferol), vitamin E (tocopherol and tocotrienols), and vitamin K (quinone). In some embodiments, the minerals are selected from sulfur, iron, chlorine, calcium, chromium, cobalt, copper, zinc, magnesium, manganese, molybdenum, iodine, and selenium.

[0052] In some embodiments, the dietary supplement is in the form of pills, powders, capsules, tablets, granular powders, opercula, orally soluble granules, sachets, sugar-coated pills, or liquids.

[0053] In some embodiments, the engineered bacteria in the dietary supplement are present in live form.

[0054] In some embodiments, the engineered bacteria in the dietary supplement are at 10 6 Up to 10 12 The amount of CFU / dose present (e.g., 10) 7 CFU / dosage, 10 8 CFU / dosage, 10 9 CFU / dosage, 10 10 CFU / dosage or 1011 CFU / dosage).

[0055] In another aspect, the present invention provides a pharmaceutical composition comprising engineered bacteria or cultures as described above.

[0056] In some embodiments, the pharmaceutical composition is a pharmaceutical composition that targets gastrointestinal release or a pharmaceutical composition that is released in a controlled manner in the gastrointestinal tract.

[0057] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0058] In some embodiments, the pharmaceutical composition further comprises additional active ingredients.

[0059] In some embodiments, the additional active ingredient is selected from anxiolytics, sedatives, hypnotics, antipsychotics, antidepressants, anticonvulsants, opioid analgesics, or any combination thereof.

[0060] In some embodiments, the pharmaceutical composition is in the form of pills, powders, capsules, tablets (e.g., effervescent tablets), film-coated tablets, orally soluble granules, liquids, suppositories, or enemas.

[0061] In another aspect, the present invention provides a method for producing equadol, the method comprising culturing engineered bacteria as described above.

[0062] In some embodiments, the method further includes recovering equadol from the culture.

[0063] In some embodiments, the method further includes adding a substance to the culture that induces equadol expression.

[0064] Studies have shown that equol, a compound produced from the metabolism of soy isoflavones, has a structure similar to estrogen and has been shown to have various potential health benefits. Furthermore, only about 30-50% of Asian populations and 20-30% of Western populations possess specific gut bacteria capable of converting daidzein into equol. These individuals are known as "equol producers." Numerous studies have shown that in postmenopausal women, the health benefits (including relief of menopausal symptoms, improved mood, and increased bone density) obtained after consuming soy are significantly stronger in "equol producers" than in "non-producers." This means that for "non-producers," directly supplementing with soy isoflavones or soy foods may have limited effects; however, supplementing with appropriate amounts of equol in the gut through engineered equol-producing probiotics can also benefit "non-producers."

[0065] Estrol has at least the following effects: (1) It exerts estrogen-like effects. For example, it can function as a selective estrogen receptor modulator (SERM); (2) Antioxidant effects. For example, it has anti-aging effects on the skin, can activate skin ERβ, promote collagen synthesis, and provide antioxidant protection for fibroblasts; for example, it protects the cardiovascular system, can inhibit LDL oxidation, and prevent atherosclerosis; (3) Anti-tumor effects. For example, it can prevent or treat prostate cancer, breast cancer, colorectal cancer, and benign prostatic hyperplasia (BPH).

[0066] Therefore, the engineered bacteria of this invention can be used in any scenario where equol exerts its effects. For example, the engineered bacteria of this invention can be used for multiple purposes such as anti-oxidation, exerting estrogen-like effects, and anti-tumor activity.

[0067] In another aspect, the present invention provides the use of engineered bacteria as described above, cultures as described above, dietary supplements as described above, or pharmaceutical compositions as described above in the preparation of pharmaceuticals for (i) preventing and / or treating mood disorders in a subject, or preventing and / or treating diseases or symptoms in a subject caused by mood disorders, (ii) preventing and / or treating diseases in a subject caused by abnormal levels of endogenous estrogen, (iii) preventing and / or treating diseases in a subject related to oxidative stress, (iv) preventing and / or treating diseases in a subject related to elevated levels of inflammation, and / or (v) preventing and / or treating tumors in a subject.

[0068] In some implementations, the mood disorder includes depression and anxiety.

[0069] In some implementations, the illness and / or symptoms caused by mood disorders (e.g., depression) are selected from: depression, bipolar disorder, seasonal affective disorder, dysphoric mood, depressive personality disorder, altered mental state, or any combination thereof.

[0070] In some implementations, the illness and / or symptoms caused by mood disorders (e.g., anxiety) are selected from: panic attacks, panic disorder, agoraphobia, social phobia or social anxiety disorder, obsessive-compulsive disorder, post-traumatic stress disorder (ASD), generalized anxiety disorder, acute stress disorder, or any combination thereof.

[0071] In some embodiments, the depression is selected from: major depressive disorder, atypical depression, typical or melancholic depression, psychotic depression, catatonic depression, prenatal and / or postnatal depression, dual depression, recurrent transient depression, mild depression, or any combination thereof.

[0072] In some embodiments, the drug is used to prevent and / or treat inflammatory diseases, prostate cancer, breast cancer, colorectal cancer, benign prostatic hyperplasia (BPH), or postmenopausal osteoporosis in a subject.

[0073] In some embodiments, the inflammatory disease is selected from diseases related to systemic inflammatory response syndrome (SIRS) (e.g., acute lung injury), diseases related to retinal inflammation (e.g., retinitis, keratitis), diseases related to skin inflammation (e.g., dermatitis, eczema), diseases related to respiratory tract inflammation (e.g., upper respiratory tract infection), and diseases related to gastrointestinal tract inflammation (e.g., inflammatory bowel disease).

[0074] Terminology Definition

[0075] 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 operational steps used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0076] As used herein, the term "exogenous" refers to nucleic acids or proteins derived from the exterior of the engineered bacteria disclosed in this invention. For example, exogenous genes can be artificially or recombinantly generated and introduced into or expressed in the engineered bacteria disclosed in this invention. Exogenous genes can also be isolated from heterologous microorganisms and introduced into or expressed in the engineered bacteria disclosed in this invention. Exogenous nucleic acids can be adapted to be integrated into the genome of the engineered bacteria disclosed in this invention or to remain in an extrachromosomal state in the engineered bacteria disclosed in this invention, for example, in plasmids.

[0077] As used herein, the term "heterologous" refers to nucleic acids or proteins not present in the wild-type microorganisms from which the engineered bacteria disclosed in this invention originate. For example, heterologous genes may be derived from different strains or species and introduced into or expressed in the engineered bacteria disclosed in this invention. Heterologous genes may be introduced into or expressed in the engineered bacteria disclosed in this invention in the form they appear in different strains or species. Alternatively, heterologous genes may be modified in some way, for example by codon optimization, for expression in the engineered bacteria disclosed in this invention.

[0078] As used herein, the term “heterologous expression” refers to the introduction of a gene encoding a protein of interest from one species or cell into another species or cell, thereby allowing the host cell to express that foreign protein.

[0079] As used herein, the term "overexpression" refers to a level of gene expression or gene product in a non-natural or recombinant microorganism that is higher than the level of gene expression or gene product found in a parent or wild-type microorganism grown under the same conditions. In some embodiments, overexpression can occur at the transcriptional, translational, or both levels, and can be due to altered regulatory controls (e.g., the use of a strong promoter) or an increase in copy number, or both. A strong promoter is a promoter with high affinity for RNA polymerase that directs the synthesis of large amounts of mRNA, functioning by efficiently initiating transcription. Common types include lacP, trpP, tacP, λPL, CaMV promoters, and adenovirus late gene promoters.

[0080] As used herein, the terms “deletion,” “knockout,” and “knockdown” have the meanings commonly understood by those skilled in the art. The term “deletion” refers to the removal or disruption of a specific gene within the genome of an organism, preventing that gene from being normally expressed, transcribed, or translated. The term “knockout” refers to the complete deletion or inactivation of the DNA sequence of a target gene from a cell or organism, blocking its transcription and translation processes, thus rendering the target gene completely nonfunctional. The deletion or knockout of endogenous genes can be achieved using any gene editing system known in the art, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or clustered regularly spaced short palindromic repeats (CRISPR). The term “knockdown” refers to reducing the expression level of a target gene in a cell or organism through gene silencing, resulting in a temporary alteration in expression. Exemplary gene silencing methods include, but are not limited to, RNA interference (RNAi), small interfering RNA (siRNA), or short hairpin RNA (shRNA), which are used to inactivate the messenger RNA of a specific gene and effectively inhibit the expression of that gene.

[0081] As used herein, the terms “wild,” “wild-type,” or “natural” are used interchangeably. When these terms are used to describe a microorganism, they mean that the microorganism exists in nature, is found in nature, and has not undergone any artificial modification or processing.

[0082] As used herein, the term "probiotic" refers to a microorganism or its cellular component that has a beneficial effect on the health or well-being of a host. Microorganisms well known in the art can be classified as probiotics; for example, see Salminen S, Ouwehand A, Benno Y, et al., "Probiotics: how should they be defined," Trends FoodSci.Technol. 1999: 10107-10; and Trends in Food Science and Technology, 1999, Vol. 10, pp. 107-110. The term "probiotic bacteria" refers to bacteria or its cellular component that has a beneficial effect on the health or well-being of a host.

[0083] As used herein, the term "dietary supplement" refers to an edible product that provides a beneficial effect to a consumer (e.g., nutritional, preventative, therapeutic, or other beneficial effect). In this document, dietary supplements encompass products such as health products, nutritional supplements, and tonics.

[0084] As used in this article, the term "CFU (Colony-Forming Units)" refers to the total number of microbial communities such as bacteria, fungi, and yeast in a product, and is usually used to calculate the number of viable cells.

[0085] As used herein, the term "CFU / dosage" refers to the amount of bacteria present in a composition / food product or dietary supplement / pharmaceutical composition provided to a subject daily or per dose. For example, in some embodiments, the engineered bacteria in the pharmaceutical composition or dietary supplement are present at a concentration of 10... 6 Up to 10 12 The amount of CFU / dose present (e.g., 10) 8 Up to 10 12 CFU / dosage). In this embodiment, if the engineered bacteria are applied to a food product (e.g., in a solid beverage, yogurt), the food product (e.g., a solid beverage, yogurt) provided to the subject daily or per dose may contain approximately 10 CFU / dosage. 6 Up to 10 12 CFU-engineered bacteria. Alternatively, the amount of these bacteria can be administered in multiple doses, provided that the total amount of engineered bacteria received by the subject at any given time (e.g., every 24-hour period) is less than approximately 10. 6 To about 10 12 CFU bacteria, i.e., engineered bacteria that meet the above-mentioned requirements in pharmaceutical compositions or dietary supplements, at a concentration of 10 6 Up to 10 12The amount of CFU / dose present (e.g., 10) 8 Up to 10 12 CFU / dosage).

[0086] As used herein, the term "pharmaceuticalally acceptable carrier" means a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19). th (ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0087] As used herein, the term "subject" refers to mammals, including but not limited to humans, rodents (mice, rats, guinea pigs), dogs, horses, cattle, cats, pigs, monkeys, chimpanzees, etc. In some embodiments, the subject is a human.

[0088] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom 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, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, relieving symptoms (whether partial or complete), alleviating or improving prognosis, reducing or inhibiting disease recurrence, whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to expected survival (if no treatment was received).

[0089] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap may be introduced in the first amino acid sequence or nucleic acid sequence to best align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecule is identical at that position. The percentage identity between two sequences is a function of the number of identity positions shared by the sequences (i.e., percentage identity = number of identical overlapping positions / total number of positions × 100%). In some embodiments, the two sequences are of the same length.

[0090] The genes disclosed in this invention are all known in the art, and their sequences are available from public databases. Furthermore, this invention provides exemplary sequences of the following genes.

[0091]

[0092] Beneficial effects

[0093] The applicant of this invention, through extensive experimentation, provides an engineered bacterium (HLE0252). Compared to bacteria modified in other ways, this engineered bacterium exhibits significantly increased equadol production and extremely high biocompatibility. Furthermore, the engineered bacterium of this invention demonstrates outstanding efficacy in improving mood disorders (e.g., anxiety, depression), effectively improving depression and anxiety in subjects (e.g., increasing mammalian curiosity about the external environment in disease models).

[0094] Therefore, the engineered bacteria of the present invention have at least the following advantages: (1) It can regulate the level of equadol production, has a clear therapeutic window, and can safely and controllably supplement equadol to subjects continuously; (2) It can improve the gut microbiota structure of the subjects, adjust the intestinal metabolic pattern of the subjects, and benefit the intestinal health of the subjects; (3) It can significantly improve the anxiety symptoms of subjects and alleviate depressive mood, or prevent negative emotions such as anxiety caused by stress (e.g., significantly improve depressive-like and anxiety-like behaviors in mice with depressive disorders induced by chronic restraint stimulation and chronic unpredictable mild stress).

[0095] 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

[0096] Figure 1 This shows a comparison of estrol production between different modified strains and the HLE0252 strain.

[0097] Figure 2 The results show the effects of different treatments on depressive-like behavior and anhedonia in mice.

[0098] (2a) Sugar water preference rate (%) in each group of mice. Compared with the control group, the model group showed a significantly reduced sugar water preference rate (P < 0.0001), indicating successful modeling; while the engineered bacteria group showed a significantly increased sugar water preference rate, suggesting that the engineered bacteria significantly improved the anhedonia in mice. (2b) Immobility time (seconds) in each group of mice during the tail suspension test. Compared with the model group, the immobility time of mice in the engineered bacteria group was significantly reduced (P < 0.01), indicating improved emotional state. (2c) Immobility time (seconds) in each group of mice during the forced swimming test. Compared with the model group, the immobility time of mice in the engineered bacteria group was significantly shortened (P < 0.05), indicating reduced anxiety level.

[0099] Figure 3 The results of the elevated cross test are displayed.

[0100] (3a) Percentage of time spent in the open arms of each group of mice in the elevated cross maze (%). Compared with the model group, the engineered bacteria group significantly prolonged the time spent in the open arms (P < 0.01), indicating a decrease in anxiety level. (3b) Number of times mice entered the open arms of each group. Compared with the model group, the engineered bacteria group significantly increased the number of times mice entered the open arms, indicating enhanced exploratory behavior and decreased anxiety-like behavior.

[0101] Figure 4 Display the open field test results.

[0102] (4a) Percentage of time spent in the central region during the open field test (%). The time spent in the central region by mice in the model group was significantly shorter than that in the control group (P < 0.01), indicating successful modeling; while the engineered bacteria group showed a certain degree of improvement compared to the model group. (4b) Total movement distance (m) of mice in the open field test. The movement distance of mice in the model group was significantly shorter than that in the control group (P < 0.01), indicating successful modeling; while the movement distance of mice in the engineered bacteria group was significantly longer than that in the model group (P < 0.01), suggesting that their autonomous activity ability and exploratory behavior were improved.

[0103] Figure 5 Display the open field test results.

[0104] (5a) Total movement distance (m) of mice in each group during the open field test. There was no significant difference in movement distance between the model group and the control group, and the movement distance of mice in the engineered bacteria group was also not significantly different from that of the model group, suggesting that the CUMS depression model did not significantly affect the motor ability of mice, and the motor levels of mice in each group were comparable. (5b) Percentage of time spent in the central area by mice in each group during the open field test (%). The central area time of mice in the model group was significantly shorter than that in the control group (P < 0.0001), indicating successful modeling; while the engineered bacteria group showed a significant improvement compared to the model group (P < 0.01), and the central area time of the chassis bacteria group was similar to that of the model group, suggesting that engineered bacteria treatment had a more significant effect on improving exploratory behavior.

[0105] Figure 6 The results of the elevated cross test are displayed.

[0106] (6a) Percentage of time spent in the open arms of each group of mice in the elevated cross maze (%). Compared with the model group, the engineered bacteria group significantly prolonged the time spent in the open arms (P < 0.001), indicating a decrease in anxiety level. (6b) Number of times mice entered the open arms of each group. Compared with the model group, the engineered bacteria group significantly increased the number of times mice entered the open arms (P < 0.01), indicating enhanced exploratory behavior and decreased anxiety-like behavior.

[0107] Figure 7 The figure shows the effects of different treatments on depressive-like behavior and anhedonia in mice with depressive disorders.

[0108] (7a) Immobility time (seconds) of mice in each group during the tail suspension test. Compared with the model group, the immobility time of mice in the engineered bacteria group was significantly reduced (P < 0.01), indicating improved emotional state. (7b) Immobility time (seconds) of mice in each group during the forced swimming test. Compared with the model group, the immobility time of mice in the engineered bacteria group was significantly shortened (P < 0.01), indicating reduced anxiety level. (7c) Sugar water preference rate (%) of mice in each group. Compared with the control group, the model group showed a significantly reduced sugar water preference rate (P < 0.0001), indicating successful modeling; while the sugar water preference rate of the engineered bacteria group reached a level similar to that of the control group, indicating a significant improvement in anhedonia caused by drug administration.

[0109] Instructions on the Preservation of Biological Materials

[0110] Escherichia coli ( Escherichia coliHLE0252 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) located on Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34947 and deposit date of June 19, 2025.

[0111] Sequence information

[0112] Information on a portion of the sequence involved in this invention is provided below.

[0113] Detailed Implementation

[0114] The invention will now be described with reference to the following examples, which are intended to illustrate the invention (and not limit it). Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are substantially in accordance with the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., A Concise Guide to Laboratory Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995.

[0115] Furthermore, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0116] Example 1. Construction of strain HLE0252

[0117] First, the target genes were obtained and ligated into the corresponding vectors. In total, the dznr, gdh, dhdr, thdr, and ddrc genes were obtained, and homologous arm sequences for ligation into the vectors were introduced into each target gene using primer amplification. Furthermore, to enable these target genes to be expressed in the vectors, partial promoter and RBS sequences were introduced into each target gene, ensuring that the target genes, after ligation into the vector, form complete promoter and RBS sequences.

[0118] This invention ligates the genes gdh and dznr into the pETDuet-1 vector, and the genes ddrc, dhdr, and thdr into the pCDFDuet vector, and transfects both vectors into chassis cells. This invention also replaces the promoter of the endogenous gene bglF in chassis cells with a strong promoter to achieve bglF overexpression. Furthermore, this invention knocks out the endogenous plasmid in chassis cells. Through the above modifications, engineered bacteria HLE0252 were obtained. The specific experimental steps are as follows.

[0119] (1) The following fragments containing the gene gdh (primers: gdh-F, gdh-R, sequences SEQ ID NO: 4 and 5), dhdr (primers: dhdr-F, dhdr-R, sequences SEQ ID NO: 10 and 11), dznr (primers: dznr-F, dznr-R, sequences SEQ ID NO: 6 and 7), thdr (primers: thdr-F, thdr-R, sequences SEQ ID NO: 12 and 13), and ddrc (primers: ddrc-F, ddrc-R, sequences SEQ ID NO: 8 and 9) were amplified by PCR from plasmid pRSFDuet-T7-dznr-gdh-dhdr-thdr-ddrc (synthesized by Suzhou Genewise Biotechnology Co., Ltd., SEQ ID NO: 1). The RBS sequence in this invention employs a segmented introduction strategy, in which 20-40 bp portions of the promoter, RBS sequence, and overlapping sequence are pre-introduced into the amplification primers for the gdh, dhdr, dznr, thdr, and ddrc fragments.

[0120] (2) To introduce the gene gdh and the gene dznr into the ampicillin-containing vector plasmid pETDuet-1, and to completely splice the target promoter sequence and RBS sequence upstream of the two genes, specific primers pETDuet-F and pETDuet-R (sequences SEQ ID NO: 14 and 15, respectively) were designed first. Using pETDuet-1 as a template, a linearized vector was obtained by PCR amplification (with overlapping sequences at both ends of the vector). At the same time, for the gdh and dznr fragments obtained in step (1), specific primers gdh-F2, gdh-R2, dznr-F2 and dznr-R2 (sequences SEQ ID NO: 16-19, respectively) with homologous sequences of the vector were designed. After two PCR amplifications, the gdh and dznr fragments obtained with complementary overlapping sequences at both ends of the linearized vector were obtained, and they were precisely docked with the remaining promoter + RBS sequence on the vector to form a complete element. Then, the linearized vector and the vector with partial promoter / RBS sequences were combined. The gdh and dznr fragments with overlapping sequences were assembled using Gibson assembly. The promoter + RBS sequence was completely spliced ​​through complementary overlapping regions, and finally the recombinant plasmid pETDuet-1-DZNR-GDH was obtained.

[0121] The sequence of the obtained vector pETDuet-1-DZNR-GDH is shown in SEQ ID NO: 2. The vector pETDuet-1-DZNR-GDH contains, from 5' to 3', the following sequence: J23119 promoter (SEQ ID NO: 35), RBS sequence (SEQ ID NO: 34), gene dznr (SEQ ID NO: 28), J23101 promoter (SEQ ID NO: 36), RBS sequence (SEQ ID NO: 34), gene gdh (SEQ ID NO: 32), and T7 terminator (SEQ ID NO: 40).

[0122] (3) Similarly, to introduce the genes ddrc, dhdr, and thdr into the streptomycin-resistant vector plasmid pCDFDuet, and to completely splice the target promoter sequence and RBS sequence upstream of the three genes, specific primers pCDFDuet-F and pCDFDuet-R (sequences SEQ ID NO: 20 and 21, respectively) were designed first. Using pCDFDuet as a template, a linearized vector was obtained by PCR amplification (with overlapping sequences at both ends of the vector). At the same time, for the ddrc, dhdr, and thdr fragments obtained in step (1), specific primers ddrc-F2, ddrc-R2, dhdr-F2, dhdr-R2, thdr-F2, and thdr-R2 (sequences SEQ ID NO: 22-27, respectively) with homologous sequences of the vector were designed. After two PCR amplifications, the ddrc, dhdr, and thdr fragments were amplified. The fragments and thdr fragments were fitted with complementary overlapping sequences at both ends to the linearized vector, and precisely docked with the remaining promoter + RBS sequence on the vector to form complete elements. Subsequently, the linearized vector, the ddrc fragment with partial promoter / RBS and overlapping sequences, the dhdr fragment and the thdr fragment were assembled by Gibson under the catalysis of a one-step cloning enzyme (ClonExpress MultiS One Step Cloning Kit, Novizan). The complete splicing of the promoter + RBS sequence was achieved through complementary overlapping regions, and finally the recombinant plasmid pCDFDuet-1-DDRC-DHDR-THDR was obtained.

[0123] The sequence of the obtained vector pCDFDuet-1-DDRC-DHDR-THDR is shown in SEQ ID NO: 3. The vector contains, from 5' to 3', the following sequences in sequence: J23100 promoter (SEQ ID NO: 37), RBS sequence (SEQ ID NO: 34), gene ddrc (SEQ ID NO: 29), J23102 promoter (SEQ ID NO: 38), RBS sequence (SEQ ID NO: 34), gene dhdr (SEQ ID NO: 30), J23104 promoter (SEQ ID NO: 39), RBS sequence (SEQ ID NO: 34), gene thdr (SEQ ID NO: 31), and T7 terminator (SEQ ID NO: 40).

[0124] (4) Knock out the two cryptic plasmids pMUT1 and pMUT2 of Escherichia coli Nissle 1917 (obtained from Beijing Bio-Bio Biotechnology Co., Ltd., catalog number: bio-089890), inoculate into 50 mL LB medium, and culture at 37°C and 220 rpm until OD. 600 Once the pH reaches 0.4-0.6, collect the bacterial culture, centrifuge at 5000 rpm for 10 min at 4℃, discard the supernatant, resuspend the precipitate with sterile ddH2O, and centrifuge at 5000 rpm for 5 min at 4℃. Repeat this step twice. Discard the supernatant, resuspend the cells with 10% glycerol and concentrate them to prepare electrocompetent cells. Store them at -80℃. Transform the recombinant plasmid pETDuet-1-DZNR-GDH and the recombinant plasmid pCDFDuet-1-DDRC-DHDR-THDR into E. coli DH5α for amplification. After confirming the correct sequence and orientation of the inserted fragments by sequencing, electroporate them into the chassis cells EcN at 2.5 kV and 5 ms to produce the recombinant strain EcN-EQL1. (5) A donor DNA fragment containing the strong promoter J23119 was designed and synthesized. The promoter of the endogenous gene bglF in the recombinant strain EcN-EQL1 was replaced with the strong promoter J23119 (SEQ ID NO: 35) through homologous recombination to construct the final strain HLE0252 of this patent. The modification information of strains EcN-EQL1 and HLE0252 is shown in Table 1, and the genes involved are shown in Table 2.

[0125] Table 1. Strain modification information

[0126] Table 2. Genetic Information

[0127] Example 2. Comparison of equadol production from different strains

[0128] In this embodiment, different modified strains were constructed and their equadol production was compared. The specific modification methods and chassis cells used are shown in Table 3.

[0129] Table 3. Modification of different strains

[0130] Note: ① Introducing external dznr, ddrc, dhdr and thdr; ②Introduce exogenous gdh; ③ Overexpression of endogenous bglF; ④ Knock out the EcN endogenous plasmid.

[0131] ⑤ Knock out ptsG (glucose transmembrane transport gene) and introduce exogenous sacC (β-fructofuranosidase).

[0132] The results are as follows Figure 1 As shown, under the same culture conditions, the equadol production of strain HLE0252 of this invention is significantly higher than that of other strains. For example, the chassis cells of the strains constructed according to methods 3 and 4 are the same as those of strain HLE0252, differing only in the adjustment of one gene. However, the equadol production of strain HLE0252 is 45.8% higher than that of the strain obtained by method 3 (i.e., EcN-EQL1 in Example 1), and as high as 331.5% higher than that of the strain obtained by method 4.

[0133] Example 3. Validation method for treating depression with strain HLE0252

[0134] This invention verifies the antidepressant therapeutic effect of the constructed engineered bacteria using two classic animal models of depression, as detailed below: Experimental animals and grouping: Healthy SPF-grade C57BL / 6 mice (7 weeks old, female) were selected and randomly divided into the following groups. All mice were acclimatized for 7 days in a standard environment (temperature 22±2℃, humidity 50±5%, 12h light-dark cycle) before the experiment began: Normal control group (WT group): No depression model was established, and the same volume of sterile PBS was given. Model control group (CRS / CUMS group): Depression model was established and the same volume of sterile PBS was administered; Chalcogenide control group (EcN group): Chalcogenide EcN (dose of 1×10⁻⁶) was administered simultaneously with the establishment of the depression model. 9 CFU / unit / day, dissolved in sterile PBS); Engineered bacteria treatment group (HLE0252 group): Depression model was established while simultaneously administering the engineered bacteria HLE0252 constructed in this invention (dosage was consistent with that of the chassis bacterium EcN, i.e., 1×10⁻⁶). 9 CFU / animal / day, dissolved in sterile PBS. In the figure, it is represented as CRS+HLE0252 or CUMS+HLE0252.

[0135] Depression Model Construction and Drug Administration: Chronic restraint stress (CRS) model: Mice in the modeling groups (CRS group, EcN group, HLE0252 group) were individually placed in restraint tubes with ventilation holes at fixed times each day (9:00-15:00). Each restraint lasted 6 hours, and the model was maintained while medication was administered for 4 consecutive weeks at a dose of 1×10⁻⁶ mg / L. 9 CFU / mouse / day; during this period, normal control mice were not restrained, but were given PBS simultaneously.

[0136] Chronic unpredictable mild stress (CUMS) model: The modeling groups (CUMS group, EcN group, HLE0252 group) were subjected to a randomized combination of mild stressors (including heat stress, empty cage exposure, vibration stress, fasting, water restriction, cage tilting, etc., with 1-2 stressors randomly selected daily to avoid predictability). Modeling and drug administration were carried out continuously for 4 weeks, with a drug dosage of 1×10 9 CFU / mouse / day; normal control mice were not subjected to any stress, but were given PBS simultaneously.

[0137] Administration method: All treatment groups were administered the drug once daily via gavage, with a volume of 0.2 mL per animal, containing a bacterial agent concentration of 5 × 10⁻⁶. 9 CFU / mL, the normal control group and the model control group were given the same volume of sterile PBS, and the process was carried out simultaneously with the modeling process for 4 weeks.

[0138] Behavioral testing and evaluation: After modeling and drug administration, the following behavioral tests were performed sequentially at 24-hour intervals to evaluate the depressive-like behavior of mice and the treatment effect: Sugar water preference test (SPT): In the adaptation phase, a single bottle is first supplied with 1% (mass fraction) sucrose solution for 24 hours to adapt to the sweetness. Then, two bottles are supplied with sucrose solution and sterile water for 24 hours, and the positions are switched every 12 hours to eliminate lateral bias (if the motivation to drink water is insufficient, water is withheld for 12 hours before the formal test). The formal test lasts for 24 hours with one switch in the middle. The sucrose intake, water intake and total intake are recorded by weighing the initial and remaining liquid. The sugar water preference rate is calculated as "sucrose intake ÷ total intake × 100%". An increase in the preference rate indicates an improvement in anhedonia-like behavior. Tail Suspension Test (TST): The tail of a mouse is fixed at 1 / 3 and suspended 50cm above the ground. The immobility time of the mouse is recorded in the last 4 minutes within 6 minutes. A shorter immobility time indicates an antidepressant effect. Forced swimming test (FST): Mice were placed in a cylindrical container with a water depth of 30 cm and a water temperature of 25 ± 1 ℃. The immobility time of the mice was recorded in the last 4 minutes of the 6-minute period. A decrease in immobility time indicated an improvement in depressive-like symptoms. Open field test (OFT): Mice are placed in an open field box measuring 50cm×50cm×30cm. The total distance the mice move (reflecting their ability to move independently) and the time spent in the central area (reflecting the improvement of anxiety-like behavior) are recorded over 5 minutes using a video tracking system. Elevated Cross Maze Test (EPM): The maze consists of two open arms (30cm×5cm) and two closed arms (30cm×5cm×15cm), with a central area of ​​5cm×5cm and 50cm above the ground. The number of times the mouse enters the open arms and the proportion of time spent in the open arms to the total test time are recorded within 5 minutes. An increase in the proportion indicates relief of anxiety-like behavior.

[0139] Example 4. Experimental study on the prevention and treatment of depressive disorders induced by chronic restraint stimulation.

[0140] 1. Objective: To observe the effects of the engineered probiotics of this invention on depressive-like behavior, activity level, and anxiety-like condition in mice with chronic restraint-induced depression, and to compare them with unmodified chassis cell Escherichia coli Nissle1917 under the same conditions.

[0141] 2. Implementation Method: C57BL / 6N normal mice were randomly assigned to a normal control group, a model group, an Escherichia coli Nissle1917 (EcN) group, and the HLE0252 treatment group of this invention for treatment and behavioral experiments. After one week of acclimatization feeding, the model group, the E. coli Nissle1917 group, and the HLE0252 group underwent chronic restraint stress, being restrained in a restraint tube for 6 hours daily. During this period, the mice were allowed to breathe freely but could not move, and this restraint continued for four weeks. The E. coli Nissle1917 group and the HLE0252 group were respectively administered E. coli Nissle1917 and estrol-producing engineered probiotic HLE0252 via intensive feeding at a dose of 2 × 10⁻⁶. 9 CFU / dose / day for four consecutive weeks. The normal control group and the model group were treated with the same volume of PBS for four consecutive weeks using a forced feeding method.

[0142] 3. Behavioral testing protocol: (1) Tail suspension test: Despair-like behavioral assessment was conducted using a mouse tail suspension device. The experimental setup consisted of a suspension bracket, fixing tape / clamps, and a video acquisition and data analysis system, and was used to detect immobile behavior in mice in situations where escape was impossible.

[0143] A) Adaptation and placement: Secure the mouse to the suspension beam with tape about 1-2 cm from the tail end, with a suspension height of about 30-50 cm, ensuring that its forelimbs are off the ground and do not touch surrounding objects.

[0144] B) Formal test: A single mouse was suspended for 6 minutes, and its immobile behavior (slight swaying with gravity and no active struggle) was recorded.

[0145] Record indicators: the time the device remains stationary within four minutes of being suspended, and the percentage of time the device remains stationary.

[0146] (2) Forced swimming test: Despair-like behaviors were assessed using a transparent cylindrical water tank. The experimental setup consisted of a water tank (approximately 10-20 cm in diameter, 15-20 cm in depth, and approximately 23-25 ​​°C in temperature) and a video acquisition and data analysis system.

[0147] A) Pretreatment: Conduct a pre-test as needed (e.g., place the tank in the tank for 10-15 minutes the day before) to reduce the effects of novelty stress.

[0148] B) Formal test: Each mouse was placed in a water tank for 6 minutes, and its behavior in the last 4 minutes was recorded.

[0149] Record metrics: cumulative immobility time, swimming / climbing (struggling) time.

[0150] (3) Sugar water preference test: The two-bottle selection paradigm was used to assess anhedonia-like behavior. The experimental setup consisted of a single-cage dual-drinking system, an electronic balance, and a data recording system.

[0151] A) Adaptation phase: Provide a 1% (mass fraction) sucrose solution for 24 hours to allow the body to adapt to the sweetness, followed by two bottles (sucrose solution and water) for 24 hours, with the left and right positions switched every 12 hours to eliminate lateral bias. If necessary, withhold water for 12 hours before the formal test.

[0152] B) Formal test: Provide sucrose solution and water simultaneously for 24 hours, weigh the liquid mass / volume before and after, and switch the liquids once.

[0153] Record indicators: sucrose intake, water intake, total intake; Sugar water preference rate = sucrose intake ÷ (sucrose + water intake) × 100%.

[0154] (4) Open field test: A square open-field box was used to assess spontaneous activity and anxiety-related behaviors. The experimental setup consisted of a bounded square box (e.g., 50 cm × 50 cm × 40 cm, with the bottom divided into a central area and a peripheral area) and a video acquisition and trajectory analysis system.

[0155] A) Adaptation and Cleanup: Each mouse was placed in a standby cage for 2-5 minutes before testing, and the cage was wiped with 75% ethanol between individuals.

[0156] B) Formal test: Gently place the mouse in a fixed corner / center of the box and test continuously for 5-10 minutes.

[0157] Record metrics: total walking distance, average speed, time spent in the central area, and number of entries.

[0158] (5) Elevated cross maze test: An elevated cross maze was used to assess anxiety-related behaviors. The experimental setup consisted of two open arms and two closed arms (perpendicular to each other, about 30-50 cm long, with no barriers on the open arms and barriers on three sides on the closed arms), an elevated platform (about 50 cm above the ground), and a video acquisition and data analysis system.

[0159] A) Adaptation and Placement: Maintain constant lighting in the laboratory (e.g., ~100-200 lx), place the mice in the central area with their heads facing any of the closed arms.

[0160] B) Formal test: Observe continuously for 5 minutes.

[0161] Record indicators: number of times the open arm is entered, dwell time in the open arm, number of times the closed arm is entered and dwell time, and dwell time in the central area.

[0162] 4. Experimental Results: (1) Effects on depression-like behavior and anhedonia in mice with depression disorder By detecting and analyzing the immobility time in the forced swimming test, the immobility time in the tail suspension test, and the sucrose preference rate in the sucrose preference test, the final results showed that the engineered bacteria of this invention could significantly improve depressive-like behavior and anhedonia in mice with depressive disorders. Specifically, the HLE0252 treatment group showed a significant decrease in immobility time in the tail suspension and forced swimming tests compared to the model group, and an increase in the sucrose preference rate in the sucrose preference test. In particular, compared to the *Escherichia coli* Nissle1917 treatment group, the sucrose preference rate in the sucrose preference test showed a better improvement. These results indicate that the engineered probiotics of this invention have the ability to significantly improve depressive-like behavior and anhedonia in mice with depressive disorders, and the therapeutic effect is significant. Experimental results are shown below. Figure 2 .

[0163] (2) Effects on anxiety-like behavior and spontaneous activity in mice with depression

[0164] Compared to the model group, the HLE0252 engineered bacteria administration group significantly increased the time mice spent in the open arm and the number of times they entered and exited the maze in the elevated cross maze test, and also effectively improved the total distance traveled in the open field test. These results indicate that the engineered bacteria have a significant effect on improving anxiety-like behavior and spontaneous activity in mice with depressive disorders. Experimental results are as follows: Figure 3 and Figure 4 As shown.

[0165] Example 5. Prevention and treatment of chronic, unpredictable, mild stress

[0166] 1. Objective: To observe the effects of the engineered probiotics of this invention on depressive-like behavior, activity level, and anxiety-like condition in mice with chronic unpredictable mild stress-induced depression, and to compare them with chassis cell Escherichia coli Nissle1917 under the same conditions.

[0167] 2. Implementation Method: C57BL / 6N normal mice were randomly assigned to a normal control group, a model group, an Escherichia coli Nissle1917 (EcN) group, and the HLE0252 treatment group of this invention for treatment and behavioral experiments. After one week of acclimatization, the model group, the E. coli Nissle1917 group, and the HLE0252 group were subjected to chronic, unpredictable, mild stress, with 1 to 2 stressors applied daily for four consecutive weeks. The E. coli Nissle1917 group and the HLE0252 group were respectively administered E. coli Nissle1917 and estrol-producing engineered probiotic HLE0252 via intensive feeding at a dose of 2 × 10⁻⁶. 9 CFU / dose / day for four consecutive weeks. The normal control group and the model group were treated with the same volume of PBS for four consecutive weeks using a forced feeding method.

[0168] 3. The following stressors should be identified: A) Inclined cage: Purpose: To disrupt the animal's balance and comfort, causing mild but persistent discomfort.

[0169] Operating procedures: Elevate one side of a standard feeding cage so that the bottom of the cage is at approximately a 45° angle to the horizontal. Keep the cage door secure to prevent the animal from falling out. Tilt the feed rack and water bottle along with the cage to ensure the animal can still reach them, but this will slightly increase the difficulty of feeding. Continue for 12 hours.

[0170] B) Damp bedding: Objective: To induce stress through uncomfortable, damp environments.

[0171] Operating steps: Add warm water to 2 / 3 of the cage volume, wetting it until it is noticeably damp but without excessive standing water. Spread the wet bedding evenly and leave it for 12 hours.

[0172] C) Swimming in cold water (4℃)

[0173] Objective: To induce transient cold stimulation and exercise stress.

[0174] Procedure: Prepare a cylindrical or rectangular container (deep enough so the animal cannot touch the bottom to prevent escape). Mix ice and water to adjust the water temperature to 4°C. Gently place the animal in the water, forcing it to swim and float. After the designated time, quickly dry it with a towel and return it to a dry, warm cage for 5 minutes.

[0175] D) Tail-clamping stress

[0176] Purpose: Transient pain stress.

[0177] Operating steps: Use a tail clip to hold the tail tip about 1 cm away for 5 minutes.

[0178] E) Day and night reversed

[0179] Objective: To disrupt biological rhythms

[0180] Operating procedures: Keep the animal room lights off from 8:00 to 20:00 to create a dark environment; keep the animal room lights on from 20:00 to 8:00 the next day to create a daytime environment.

[0181] F) Food deprivation

[0182] Objective: Starvation stress

[0183] Operating procedures: At 8:00 AM, remove all feed from the cage, leaving only water. Resume feeding on schedule 24 hours later.

[0184] G) Deprivation of drinking water

[0185] Objective: Thirst stress

[0186] Operating procedures: At 8:00 AM, remove all water from the cages, leaving only feed. Restore water access 24 hours later.

[0187] H) Vibration stress

[0188] Objective: To induce restlessness and mild physical stress through continuous, slight vibrations.

[0189] Operating procedures: Place the animal, cage and all, on the vibrating plate. Set to low-frequency, gentle vibration (80 rpm) and provide continuous stimulation for 20 minutes.

[0190] I) Empty cage exposed (no bedding, no sheltered environment)

[0191] Purpose: To deprive individuals of potential comfort and security, which constitutes mild environmental stress.

[0192] Procedure: Transfer the animal to a clean, empty cage without bedding or nesting material. Leave the water bottle on for 12 hours.

[0193] J) Thermal high-temperature stress

[0194] Objective: To induce physical stress through appropriate high-temperature stimulation.

[0195] Procedure: Transfer the animal to an environment of 42°C for 20 minutes.

[0196] During implementation, the aforementioned stressors should be administered at most once a week, without repetition.

[0197] 4. Behavioral testing protocol: 1) Tail suspension test: Despair-like behavioral assessment was conducted using a mouse tail suspension device. The experimental setup consisted of a suspension bracket, fixing tape / clamps, and a video acquisition and data analysis system, and was used to detect immobile behavior in mice in situations where escape was impossible.

[0198] A) Adaptation and placement: Secure the mouse to the suspension beam with tape about 1-2 cm from the tail end, with a suspension height of about 30-50 cm, ensuring that its forelimbs are off the ground and do not touch surrounding objects.

[0199] B) Formal test: A single mouse was suspended for 6 minutes, and its immobile behavior (slight swaying with gravity and no active struggle) was recorded.

[0200] Record indicators: the time the device remains stationary within four minutes of being suspended, and the percentage of time the device remains stationary.

[0201] 2) Forced swimming test: Despair-like behaviors were assessed using a transparent cylindrical water tank. The experimental setup consisted of a water tank (approximately 10-20 cm in diameter, 15-20 cm in depth, and approximately 23-25 ​​°C in temperature) and a video acquisition and data analysis system.

[0202] A) Pretreatment: Conduct a pre-test as needed (e.g., place the tank in the tank for 10-15 minutes the day before) to reduce the effects of novelty stress.

[0203] B) Formal test: Each mouse was placed in a water tank for 6 minutes, and its behavior in the last 4 minutes was recorded.

[0204] Record metrics: cumulative immobility time, swimming / climbing (struggling) time.

[0205] 3) Sugar water preference test: The two-bottle selection paradigm was used to assess anhedonia-like behavior. The experimental setup consisted of a single-cage dual-drinking system, an electronic balance, and a data recording system.

[0206] A) Adaptation phase: Provide a 1% (mass fraction) sucrose solution for 24 hours to allow the body to adapt to the sweetness, followed by two bottles (sucrose solution and water) for 24 hours, with the left and right positions switched every 12 hours to eliminate lateral bias. If necessary, withhold water for 12 hours before the formal test.

[0207] B) Formal test: Provide sucrose solution and water simultaneously for 24 hours, weigh the liquid mass / volume before and after, and switch the liquids once.

[0208] Record indicators: sucrose intake, water intake, total intake; Sugar water preference rate = sucrose intake ÷ (sucrose + water intake) × 100%.

[0209] 4) Open field test: A square open-field box was used to assess spontaneous activity and anxiety-related behaviors. The experimental setup consisted of a bounded square box (e.g., 50 cm × 50 cm × 40 cm, with the bottom divided into a central area and a peripheral area) and a video acquisition and trajectory analysis system.

[0210] A) Adaptation and Cleanup: Each mouse was placed in a standby cage for 2-5 minutes before testing, and the cage was wiped with 75% ethanol between individuals.

[0211] B) Formal test: Gently place the mouse in a fixed corner / center of the box and test continuously for 5-10 minutes.

[0212] Record metrics: total walking distance, average speed, time spent in the central area, and number of entries.

[0213] 5) Elevated cross maze test: An elevated cross maze was used to assess anxiety-related behaviors. The experimental setup consisted of two open arms and two closed arms (perpendicular to each other, about 30-50 cm long, with no barriers on the open arms and barriers on three sides on the closed arms), an elevated platform (about 50 cm above the ground), and a video acquisition and data analysis system.

[0214] A) Adaptation and Placement: Maintain constant lighting in the laboratory (e.g., ~100-200 lx), place the mice in the central area with their heads facing any of the closed arms.

[0215] B) Formal test: Observe continuously for 5 minutes.

[0216] Record indicators: number of times the open arm is entered, dwell time in the open arm, number of times the closed arm is entered and dwell time, and dwell time in the central area.

[0217] 5. Experimental Results: 1) Effects on anxiety-like behavior and spontaneous activity in mice with depression Anxiety status and spontaneous motor ability of mice in each group were assessed using the elevated cross test (EPM) and open field test (OFT). Regarding anxiety-like behaviors: the elevated cross test results showed ( Figure 6 (a, 6b) Compared with the WT group, the percentage of time spent and the number of times mice entered the open arm in the CUMS model group were significantly reduced (p < 0.05), indicating that the model group produced obvious anxiety-like behavior. After intervention with the engineered bacteria (HLE0252) of this invention, the time spent in the open arm and the number of times mice entered were significantly improved compared with the CUMS group (p < 0.01 or p < 0.001), while no significant improvement was observed in the EcN group. Open field test results ( Figure 5 b) This trend was further verified; the CUMS group mice spent significantly less time in the central region than the WT group (p < 0.0001), while the intervention of this invention (HLE0252) significantly increased their exploration time in the central region (p < 0.01). Regarding spontaneous activity ability: the total distance traveled in the open field test showed ( Figure 5 a) There was no significant pathological difference (ns) in the total distance moved by mice in each group, indicating that the motor function of mice in each group was normal, and the interference of motor disorders on the anxiety test results was excluded.

[0218] 2) Effects on depressive-like behavior and anhedonia in mice with depressive disorders

[0219] Despair behavior in mice was assessed using the tail suspension test (TST) and forced swimming test (FST), while anhedonia was assessed using the sucrose preference test (SPT). (1) Improvement in despair behavior: Experimental results showed that ( Figure 7 (a and 7b) Compared with the WT group, the immobility time of mice in the CUMS model group was significantly increased in both the tail suspension test and the forced swimming test (p < 0.001), indicating that the mice in the model group exhibited obvious depressive-like hopelessness behavior. After intervention with the engineered bacteria of this invention, the immobility time of mice in the TST and FST was significantly shortened compared with the CUMS group (p < 0.01), effectively alleviating depressive-like symptoms. In contrast, the EcN intervention group did not show significant improvement in either of these two tests (ns), proving that the HLE0252 of this invention has significantly stronger antidepressant activity. (2) Relief of anhedonia: The results of the sucrose preference test showed that ( Figure 7 c) The sucrose preference rate in the CUMS group was significantly lower than that in the WT group (p < 0.0001), exhibiting typical anhedonia characteristics. After intervention with the present invention (HLE0252), the sucrose preference rate of the mice was significantly restored (p < 0.0001), and the degree of restoration was close to that of the WT group. Although the EcN group also showed some improvement (p < 0.05), its effect was significantly weaker than that of the HLE0252 group.

[0220] 6. Experimental Conclusions

[0221] In summary, compared with existing technologies, this invention creatively utilizes synthetic biology principles and gene editing technology to develop an engineered probiotic, providing a new treatment modality for depressive disorders. Experimental animal studies have demonstrated that the strain HLE0252 of this invention has a significantly higher equol production, effectively and controllably supplementing the host with equol. Behavioral tests have also shown that the engineered strain developed in this invention has a good synergistic effect in repairing anhedonia, alleviating hopelessness, and combating typical depressive symptoms of anxiety. This provides a highly innovative and potentially applicable technical solution for the prevention and treatment of depression and related disorders. Furthermore, no toxic side effects have been found with this invention, and it can be used as a nutritional therapeutic food alongside other medications, offering advantages such as speed, convenience, and low cost. It has significant social benefits and is worthy of widespread promotion.

[0222] 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. An engineered bacterium, said engineered bacterium differing from its wild-type derivative in at least the following aspects: (1) Overexpress the following genes: daidzein reductase gene dznr, dihydrodaidzein racemase gene ddrc, dihydrodaidzein reductase gene dhdr, tetrahydrodaidzein reductase gene thdr, glucose dehydrogenase gene gdh, and beta-glucoside permease gene bglF; (2) Low expression or no expression: the endogenous plasmid of the wild-type microorganism.

2. The engineered bacteria according to claim 1, wherein, The bacteria in question are probiotics; Preferably, the bacteria is Escherichia coli; Preferably, the wild-type microorganism is Escherichia coli Nissle 1917 (EcN). Preferably, the endogenous plasmids of the wild-type microorganism include pMUT1 and / or pMUT2; Preferably, the engineered bacteria do not express the endogenous plasmids pMUT1 and pMUT2 of the EcN.

3. The engineered bacteria according to claim 1 or 2, wherein, Gene overexpression can be either the overexpression of an endogenous gene or the heterologous expression of an exogenous gene. Preferably, the engineered bacteria, compared to the wild-type microorganism from which it originated, contain heterologous expression of exogenous dznr, ddrc, dhdr, thdr and / or gdh; Preferably, the engineered bacteria contain endogenous overexpression of bglF compared to the wild-type microorganism from which it originated.

4. The engineered bacteria according to any one of claims 1-3, wherein, The engineered bacteria have one or more of the following technical features: (1) Includes heterologous expression of dznr derived from Slackia isoflavoniconvertens; (2) Includes heterologous expression of ddrc derived from Slackia isoflavoniconvertens; (3) Includes heterologous expression of dhdr derived from transformed isoflavoniconvertens; (4) Includes heterologous expression of thdr derived from transformed isoflavoniconvertens; (5) Includes heterologous expression of gdh derived from Priestia megaterium; (6) Use a strong promoter to drive endogenous overexpression of bglF; Preferably, the strong promoter comprises or has a nucleotide sequence as shown in SEQ ID NO:

35.

5. The engineered bacteria according to any one of claims 1-4, wherein, The engineered bacteria have one or more of the following technical features: (1) The dznr includes or has a nucleotide sequence as shown in SEQ ID NO: 28, or a nucleotide sequence having at least 80% sequence identity with it; (2) The ddrc includes or has a nucleotide sequence as shown in SEQ ID NO: 29, or a nucleotide sequence having at least 80% sequence identity with it; (3) The dhdr includes or has a nucleotide sequence as shown in SEQ ID NO: 30, or a nucleotide sequence having at least 80% sequence identity with it; (4) The thdr includes or has a nucleotide sequence as shown in SEQ ID NO: 31, or a nucleotide sequence having at least 80% sequence identity with it; (5) The gdh includes or has a nucleotide sequence as shown in SEQ ID NO: 32, or a nucleotide sequence having at least 80% sequence identity with it; (6) The bglF includes or has a nucleotide sequence as shown in SEQ ID NO: 33, or a nucleotide sequence having at least 80% sequence identity with it.

6. The engineered bacteria according to any one of claims 1-5, wherein, The engineered bacteria is Escherichia coli; Preferably, the engineered bacteria are deposited at the China General Microbiological Culture Collection Center (CGMCC) and have accession number CGMCC No. 34947.

7. A culture comprising the engineered bacteria according to any one of claims 1-6; Preferably, the culture contains at least 50 mg / L, at least 80 mg / L, at least 120 mg / L, at least 200 mg / L, at least 300 mg / L, at least 400 mg / L, or at least 500 mg / L equadol.

8. A dietary supplement comprising the engineered bacteria of any one of claims 1-6 or the culture of claim 7; Preferably, the dietary supplement is formulated for oral administration; Preferably, the dietary supplement further comprises prebiotics; Preferably, the dietary supplement further comprises melatonin, omega-3 dietary supplements, or any combination thereof; Preferably, the dietary supplement is in the form of pills, powders, capsules, tablets, granular powders, opercula, orally soluble granules, sachets, sugar-coated pills, or liquids. Preferably, the engineered bacteria in the dietary supplement are in live form; Preferably, the engineered bacteria in the dietary supplement are at a concentration of 10... 6 Up to 10 12 The amount of CFU / dose present (e.g., 10) 7 CFU / dosage, 10 8 CFU / dosage, 10 9 CFU / dosage, 10 10 CFU / dosage or 10 11 CFU / dosage).

9. A pharmaceutical composition comprising the engineered bacteria according to any one of claims 1-6 or the culture according to claim 7; Preferably, the pharmaceutical composition is a pharmaceutical composition that targets gastrointestinal release or a pharmaceutical composition that is released in a controlled manner in the gastrointestinal tract; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier; Preferably, the pharmaceutical composition further comprises additional active ingredients; Preferably, the additional active ingredient is selected from anxiolytics, sedatives, hypnotics, antipsychotics, antidepressants, anticonvulsants, opioid analgesics, or any combination thereof; Preferably, the pharmaceutical composition is in the form of pills, powders, capsules, tablets (e.g., effervescent tablets), film-coated tablets, orally soluble granules, liquids, suppositories, or enemas.

10. A method for producing estrol, the method comprising culturing the engineered bacteria according to any one of claims 1-6; Optionally, the method further includes recovering equadol from the culture; Preferably, the method further includes adding a substance that induces equadol expression to the culture.

11. Use of the engineered bacteria of any one of claims 1-6, the culture of claim 7, the dietary supplement of claim 8, or the pharmaceutical composition of claim 9 in the preparation of a pharmaceutical product, wherein the pharmaceutical product is used for (i) prevention and / or treatment of mood disorders in a subject, or prevention and / or treatment of diseases or symptoms caused by mood disorders in a subject, (ii) prevention and / or treatment of diseases in a subject caused by abnormal levels of endogenous estrogen, (iii) prevention and / or treatment of diseases in a subject related to oxidative stress, (iv) prevention and / or treatment of diseases in a subject related to elevated levels of inflammation, and / or (v) prevention and / or treatment of tumors in a subject; Preferably, the mood disorder includes depression and anxiety; Preferably, the illness or symptoms caused by mood disorders (e.g., depression) are selected from: depression, bipolar disorder, seasonal affective disorder, dysphoric mood, depressive personality disorder, altered mental state, or any combination thereof; Preferably, the illness or symptoms caused by the mood disorder (e.g., anxiety) are selected from: panic attacks, panic disorder, agoraphobia, social phobia or social anxiety disorder, obsessive-compulsive disorder, post-traumatic stress disorder (ASD), generalized anxiety disorder, acute stress disorder, or any combination thereof; Preferably, the depression is selected from: major depressive disorder, atypical depression, typical or melancholic depression, psychotic depression, catatonic depression, prenatal and / or postnatal depression, dual depression, recurrent transient depression, mild depression, or any combination thereof. Preferably, the drug is used to prevent and / or treat inflammatory diseases, prostate cancer, breast cancer, colorectal cancer, benign prostatic hyperplasia (BPH), or postmenopausal osteoporosis in the subject.

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