Engineered yeast, kits containing the engineered yeast, and uses thereof

Abstract

The present application belongs to the field of biotechnology, and particularly relates to an engineered yeast, a kit containing the same and use thereof, the engineered yeast comprising: a chassis yeast cell, the genome of which is integrated with a reporter gene expression unit regulated by a transcriptional activator protein; and a chimeric receptor combination expressed on the surface of the chassis yeast cell. The engineered yeast of the present application is obtained by synthetic biology modification, and is constructed to obtain an integrated engineered yeast platform based on a chassis yeast cell, comprising a chimeric receptor combination and other functional modules. In specific applications, the engineered yeast is delivered orally and reaches the intestinal tract, can specifically perceive target inflammatory factors in situ in the local microenvironment of the intestinal tract, and convert the biological recognition event into detectable (such as fluorescent) intracellular gene expression of the engineered yeast, thereby realizing non-invasive, real-time, high-sensitivity dynamic monitoring of the intestinal microenvironment of the subject in terms of the level of inflammatory factors.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to engineered yeast, reagent kits containing the engineered yeast, and their uses. Background Technology

[0002] Inflammatory bowel disease (IBD) is a complex chronic intestinal disease. Its symptoms mainly include diarrhea, mucus and bloody stools, abdominal pain, and abdominal distension. In severe cases, it can even lead to serious complications such as massive rectal bleeding, intestinal stricture, intestinal perforation, and toxic megacolon. Currently, clinical detection of IBD mainly includes several methods such as endoscopy, imaging assessment, blood biomarkers, and fecal biomarkers. However, these methods all have significant limitations, as follows: (1) Although endoscopy is accurate, it is invasive, complex to operate, and expensive to detect, making it inconvenient for early screening and dynamic monitoring of IBD; (2) Traditional imaging assessment carries a certain risk of ionizing radiation; (3) Blood biomarkers, such as C-reactive protein, are difficult to accurately reflect the level of local intestinal inflammation, and their sensitivity and specificity are insufficient; (4) Fecal biomarkers, such as calprotectin, are easily affected by sample quality and can only provide static inflammatory information, making it impossible to achieve real-time, in vivo dynamic monitoring.

[0003] As research into inflammatory bowel disease (IBD) deepens, studies have confirmed that inflammatory factors IL-1β and IL-17 are significantly elevated in the intestinal tissue, intestinal contents, and fecal samples of IBD patients. This phenomenon is not significant in patients with other common intestinal inflammations such as infectious enteritis. This suggests that early screening and dynamic monitoring of IBD can be achieved by sensing and specifically responding to changes in the concentration of IL-1β and IL-17 in situ at intestinal lesions.

[0004] Engineered microorganisms such as yeast have been widely explored as delivery carriers for in vivo biosensors or therapeutic molecules due to their oral safety, ability to survive in the gastrointestinal environment, ease of separation and recovery (which can be recovered from feces), and milder effects on the host compared to bacteria.

[0005] Therefore, there is an urgent need to develop a novel detection platform that can non-invasively, in real time, dynamically and specifically reflect the levels of local intestinal inflammatory factors IL-1β and IL-17, which is of great significance for the early screening and dynamic monitoring of inflammatory bowel disease. Summary of the Invention

[0006] This invention aims to at least partially address one of the technical problems existing in the prior art. To this end, this invention provides engineered yeast, a kit containing the engineered yeast, and its uses. The engineered yeast of this invention, through synthetic biology modification, constructs an integrated engineered yeast platform based on chassis yeast cells and including functional modules such as chimeric receptor combinations. In specific applications, this engineered yeast, administered orally, reaches the intestine and can in situ and specifically sense target inflammatory factors in the local intestinal microenvironment. It converts this biorecognition event into detectable (e.g., fluorescence) gene expression within the engineered yeast cells, thereby achieving non-invasive, real-time, and highly sensitive dynamic monitoring of inflammatory factor levels in the subject's intestinal microenvironment, with broad application prospects.

[0007] In a first aspect, the present invention provides an engineered yeast. According to an embodiment of the present invention, the engineered yeast comprises: a chassis yeast cell whose genome integrates a reporter gene expression unit regulated by a transcriptional activating protein; and a chimeric receptor assembly expressed on the surface of the chassis yeast cell; wherein the chimeric receptor assembly comprises at least one set of chimeric receptor pairs, each set of the chimeric receptor pairs comprising a first chimeric receptor and a second chimeric receptor; both the first chimeric receptor and the second chimeric receptor contain an extracellular region, a transmembrane region, and an intracellular region, wherein the transmembrane region is connected to the extracellular region, and the intracellular region is connected to the transmembrane region; the extracellular region of the first chimeric receptor can bind to inflammatory factors. Heterogeneous binding; the intracellular region of the first chimeric receptor contains a protease catalytic domain; the extracellular region of the second chimeric receptor can specifically recognize and bind to the complex formed by the inflammatory factor and the extracellular region of the first chimeric receptor, thereby causing dimerization of the first chimeric receptor and the second chimeric receptor; the intracellular region of the second chimeric receptor includes a cleavage recognition sequence of the protease and the transcriptional activating protein linked to its C-terminus; the dimerization causes the protease catalytic domain to cleave the cleavage recognition sequence to release the transcriptional activating protein into the cell nucleus, thereby activating the reporter gene expression unit. According to embodiments of the present invention, the engineered yeast is modified through synthetic biology to construct an integrated engineered yeast platform based on chassis yeast cells and including functional modules such as chimeric receptor combinations. Specifically, the engineered yeast of the present invention, when orally administered and reaching the intestine, specifically recognizes and binds to target inflammatory factors through the combined action of the first and second chimeric receptors on its cell surface. This induces receptor dimerization events to precisely trigger pre-installed intracellular protease cleavage molecular switches, thereby releasing transcriptional activating proteins and ultimately driving the controllable expression of reporter gene expression units. This design enables non-invasive, real-time, and highly sensitive dynamic monitoring of inflammatory factor levels in the intestinal microenvironment of subjects and / or treatment of intestinal diseases, with broad application prospects.

[0008] According to embodiments of the present invention, the engineered yeast described above may also have at least one of the following additional technical features: According to embodiments of the present invention, the transcriptional activating protein includes one or more of rtTA, GAL4, Cat8, Sip4, Aca1, and Aca2.

[0009] According to an embodiment of the present invention, the transcriptional activating protein is a tetracycline reverse transcription activating protein (rtTA), and the reporter gene expression unit contains a tetracycline response element.

[0010] According to an embodiment of the present invention, the inflammatory factor is IL-1β or IL-17.

[0011] According to embodiments of the present invention, the protease catalytic domain includes one or more of the following: tobacco etch virus protease catalytic domain, SUMO protease catalytic domain, Atg4p protease catalytic domain, HRV 3C protease catalytic domain, and NEDP1 protease catalytic domain.

[0012] According to an embodiment of the present invention, the protease catalytic domain is the tobacco etch virus protease catalytic domain.

[0013] According to an embodiment of the present invention, the protease catalytic domain is the tobacco etch virus protease catalytic domain, and the amino acid sequence of the cleavage recognition sequence is shown in SEQ ID NO: 1.

[0014] According to an embodiment of the present invention, the extracellular region of the first chimeric receptor is the extracellular domain of the IL-1R1 protein, and the extracellular region of the second chimeric receptor is the extracellular domain of the IL-1RAcP protein.

[0015] According to an embodiment of the present invention, the extracellular region of the first chimeric receptor is the extracellular domain of the IL-17RA protein, and the extracellular region of the second chimeric receptor is the extracellular domain of the IL-17RC protein.

[0016] According to an embodiment of the present invention, the reporter gene expression unit further includes the coding sequence of a fluorescently labeled protein.

[0017] According to an embodiment of the present invention, the genome of the chassis yeast cell further integrates genes encoding the first chimeric receptor and the second chimeric receptor.

[0018] According to an embodiment of the present invention, the transmembrane regions of the first chimeric receptor and the second chimeric receptor are both derived from membrane proteins of yeast cells.

[0019] In a second aspect, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises: the engineered yeast described in the first aspect.

[0020] Those skilled in the art will understand that the features and advantages described above for engineered yeast also apply to this kit, and will not be repeated here.

[0021] In a third aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises the engineered yeast described in the first aspect. According to embodiments of the present invention, after oral administration to the intestine, the pharmaceutical composition containing the aforementioned engineered yeast is specifically activated and released only in the pathological microenvironment where the concentration of local inflammatory factors is elevated, thereby achieving lesion-targeted, on-demand drug delivery, further improving the precision and low-side-effect treatment of intestinal diseases (such as inflammatory bowel disease).

[0022] In a fourth aspect of the invention, the use of the engineered yeast described in the first aspect in the preparation of reagents or kits for the detection of inflammatory bowel disease is proposed.

[0023] Those skilled in the art will understand that the features and advantages described above for engineered yeast also apply to this application, and will not be repeated here.

[0024] According to embodiments of the present invention, the above-described uses may also have the following additional technical features: According to an embodiment of the present invention, the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

[0025] In a fifth aspect of the invention, the use of the engineered yeast described in the first aspect in the preparation of reagents or kits for detecting inflammatory factors is proposed.

[0026] Those skilled in the art will understand that the features and advantages described above for engineered yeast also apply to this application, and will not be repeated here.

[0027] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0028] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 The graph shows the sensitivity test results of engineered yeast sensing strain 1 in Example 1 of the present invention. In this graph, A is the IL-1β sensitivity test result of control group 1, B is the IL-1β sensitivity test result of control group 2, C is the IL-1β sensitivity test result of control group 3, and D is the IL-1β sensitivity test result of experimental group. Figure 2The graph shows the sensitivity test results of the engineered yeast sensing strain 2 in Example 1 of the present invention. In the graph, A is the IL-17 sensitivity test result of control group 1, B is the IL-17 sensitivity test result of control group 2, C is the IL-17 sensitivity test result of control group 3, and D is the IL-17 sensitivity test result of the experimental group. Figure 3 The images show the GFP fluorescence intensity detection results of yeast cells in fecal samples from each group in Example 2 of the present invention. In this image, A shows the GFP fluorescence intensity detection results of yeast cells in fecal samples from control group 1 / experimental group 1, and B shows the GFP fluorescence intensity detection results of yeast cells in fecal samples from control group 2 / experimental group 2. Detailed Implementation

[0029] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0030] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0031] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0032] In this document, the terms “comprising” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0033] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0034] Terms and Definitions In this paper, the term "engineered yeast" refers to yeast strains that are not naturally occurring and have specific functions, obtained by genetically engineering yeast cells (such as Saccharomyces cerevisiae) to integrate exogenous functional elements (such as regulated reporter gene expression units, chimeric receptor genes, etc.) into their genome.

[0035] In this paper, the term "chassis yeast cell" refers to the yeast cell on which the modification was based, whose genome has been stably integrated with a reporter gene expression unit regulated by a transcriptional activating protein. This unit is in a strictly controlled state, normally turned off, and only turned on by the regulation of the transcriptional activating protein.

[0036] In this paper, the term "transcription activator protein" refers to a protein that can bind to a specific DNA sequence and activate the transcription of downstream genes, such as tetracycline transactivator protein.

[0037] In this document, the term "reporter gene expression unit" refers to a DNA sequence containing a promoter / regulatory element, a reporter gene (such as a fluorescent reporter gene), and a terminator, the expression of which in the engineered yeast of this invention is regulated by the transcriptional activating protein.

[0038] In this document, the term "chimeric receptor combo" refers to a group of transmembrane proteins that are artificially designed and constructed and co-expressed on the surface of engineered yeast cells of the present invention, including at least a pair of first chimeric receptors and second chimeric receptors, which work together to recognize and transduce target inflammatory factors.

[0039] In this document, the term "dimerization" refers to the process in which, during the practical application of the engineered yeast of the present invention, the extracellular region of a first chimeric receptor on its surface specifically binds to an inflammatory factor, and the extracellular region of a second chimeric receptor specifically recognizes and binds to the complex formed by the inflammatory factor and the extracellular region of the first chimeric receptor, causing the two chimeric receptors to approach each other spatially and form a stable complex.

[0040] In this document, the term "protease catalytic domain" refers to a portion of the engineered yeast of the present invention that has specific protease activity, located in the intracellular region of the first chimeric receptor, and whose function is to cleave the specific cleavage recognition sequence.

[0041] engineered yeast This invention proposes an engineered yeast. According to an embodiment of the invention, the engineered yeast comprises: a chassis yeast cell whose genome integrates a reporter gene expression unit regulated by a transcriptional activating protein; and a chimeric receptor assembly expressed on the surface of the chassis yeast cell; wherein the chimeric receptor assembly comprises at least one set of chimeric receptor pairs, each set of chimeric receptor pairs comprising a first chimeric receptor and a second chimeric receptor; both the first chimeric receptor and the second chimeric receptor contain an extracellular region, a transmembrane region, and an intracellular region, wherein the transmembrane region is connected to the extracellular region, and the intracellular region is connected to the transmembrane region; the extracellular region of the first chimeric receptor can bind to inflammatory factors. Heterogeneous binding; the intracellular region of the first chimeric receptor contains a protease catalytic domain; the extracellular region of the second chimeric receptor can specifically recognize and bind to the complex formed by the inflammatory factor and the extracellular region of the first chimeric receptor, thereby causing dimerization of the first chimeric receptor and the second chimeric receptor; the intracellular region of the second chimeric receptor includes a cleavage recognition sequence of the protease and the transcriptional activating protein linked to its C-terminus; the dimerization causes the protease catalytic domain to cleave the cleavage recognition sequence to release the transcriptional activating protein into the cell nucleus, thereby activating the reporter gene expression unit. According to embodiments of the present invention, the engineered yeast is modified through synthetic biology to construct an integrated engineered yeast platform based on chassis yeast cells and including functional modules such as chimeric receptor combinations. Specifically, the engineered yeast of the present invention, when orally administered and reaching the intestine, specifically recognizes and binds to target inflammatory factors through the combined action of the first and second chimeric receptors on its cell surface. This induces receptor dimerization events to precisely trigger pre-installed intracellular protease cleavage molecular switches, thereby releasing transcriptional activating proteins and ultimately driving the controllable expression of reporter gene expression units. This design enables non-invasive, real-time, and highly sensitive dynamic monitoring of inflammatory factor levels in the intestinal microenvironment of subjects and / or treatment of intestinal diseases, with broad application prospects.

[0042] It is understood that in the phrase "the chimeric receptor combination includes at least one set of chimeric receptor pairs, and each set of chimeric receptor pairs includes a first chimeric receptor and a second chimeric receptor," the "chimeric receptor combination" is not limited to a single functional unit, but covers at least one set of chimeric receptor pairs. Each set of chimeric receptor pairs consists of a first chimeric receptor and a second chimeric receptor paired together. The inflammatory factors specifically recognized by different sets of chimeric receptor pairs may be the same or different. Each set of chimeric receptor pairs independently follows the signal transduction mechanism of "target inflammatory factor binding → receptor dimerization → protease cleavage → transcriptional activation protein release → reporter gene activation." This design enables engineered yeast to simultaneously monitor multiple inflammatory factors, thereby achieving more accurate monitoring of inflammatory states.

[0043] It should be noted that the "chassis yeast cell" described in this invention is not limited to a specific species or strain. Any yeast cell that can serve as a synthetic biology modification platform, is compatible with the reporter gene expression unit and chimeric receptor combination, and can reach the intestinal environment after oral administration to achieve the function of this invention, including but not limited to Saccharomyces cerevisiae, Pichia pastoris, Pichia pastoris, and Candida albicans, is within the scope of protection of this invention. Those skilled in the art can select appropriate yeast species or strains as chassis cells based on different requirements for growth characteristics, ease of genetic manipulation, protein expression and secretion capabilities, gastrointestinal tolerance, and safety. In this embodiment of the invention, the inventors use Saccharomyces cerevisiae, which has a clear genetic background, a mature operating system, high safety, and a history of food-grade application, as an example for illustrative purposes.

[0044] According to embodiments of the present invention, the transcriptional activating protein includes one or more of rtTA, GAL4, Cat8, Sip4, Aca1, and Aca2.

[0045] It should be noted that the specific type of transcription activating protein in the engineered yeast of the present invention is not strictly limited. As long as it can be cleaved and released by the protease, enter the cell nucleus, and specifically bind to the response element in the reporter gene expression unit to activate the expression of downstream genes, it can be used to realize the present invention. Therefore, those skilled in the art can select appropriate transcription activating proteins according to their needs, and all of them are within the protection scope of the present invention.

[0046] According to a preferred embodiment of the present invention, the transcriptional activating protein is tetracycline reverse transcription activator (rtTA), and the reporter gene expression unit contains a tetracycline response element. Thus, by further optimizing the selection and introduction of tetracycline reverse transcription activator (rtTA) and its corresponding tetracycline response element (TetO) into the engineered yeast of the present invention, accurate regulation of the reporter gene expression unit can be achieved.

[0047] According to an embodiment of the present invention, the inflammatory factor is IL-1β or IL-17. Thus, by adjusting the first chimeric receptor and the second chimeric receptor of the engineered yeast of the present invention, it can accurately identify different inflammatory factors. Taking IL-1β or IL-17 as examples, these are two inflammatory factors that are significantly elevated in the intestinal tissue, intestinal contents, and fecal samples of patients with inflammatory bowel disease, but not significantly elevated in the intestinal tissue, intestinal contents, and fecal samples of patients with other common intestinal inflammations. Therefore, when the engineered yeast of the present invention is used in combination (with the target inflammatory factors being IL-1β and IL-17, respectively), it can be used for non-invasive, real-time, and dynamic monitoring of inflammatory bowel disease.

[0048] According to embodiments of the present invention, the protease catalytic domain includes one or more of the following: tobacco etch virus protease catalytic domain, SUMO protease catalytic domain, Atg4p protease catalytic domain, HRV 3C protease catalytic domain, and NEDP1 protease catalytic domain.

[0049] It should be noted that the engineered yeast of the present invention does not strictly limit the specific type of the protease catalytic domain. As long as it can pair with the corresponding cleavage recognition sequence and specifically cleave the recognition sequence when the first and second chimeric receptors dimerize, thereby effectively releasing the transcriptional activating protein, it can be used to realize the present invention. Therefore, those skilled in the art can select other suitable protease catalytic domains according to the requirements of cleavage efficiency and biocompatibility, all of which are within the protection scope of the present invention.

[0050] According to a preferred embodiment of the present invention, the protease catalytic domain is the tobacco etch virus protease catalytic domain.

[0051] According to an embodiment of the present invention, the protease catalytic domain is the tobacco etched virus protease catalytic domain, and the amino acid sequence of the cleavage recognition sequence is shown in SEQ ID NO: 1. Therefore, by further optimizing the selection of the tobacco etched virus protease catalytic domain and its corresponding cleavage recognition sequence in the engineered yeast of the present invention, accurate regulation of the reporter gene expression unit can be achieved.

[0052] ENLYFQG (SEQ ID NO: 1) According to an embodiment of the present invention, the extracellular region of the first chimeric receptor is the extracellular domain of the IL-1R1 protein, and the extracellular region of the second chimeric receptor is the extracellular domain of the IL-1RAcP protein.

[0053] According to an embodiment of the present invention, the extracellular region of the first chimeric receptor is the extracellular domain of the IL-17RA protein, and the extracellular region of the second chimeric receptor is the extracellular domain of the IL-17RC protein.

[0054] It should be noted that although the embodiments of the present invention only use mouse receptors as an example for verification, based on the high homology of IL-1R1 / IL-1RAcP and IL-17RA / IL-17RC among different mammals, those skilled in the art can reasonably expect that human and other mammalian receptors will also be able to achieve the corresponding functions.

[0055] According to an embodiment of the present invention, the reporter gene expression unit further includes a coding sequence for a fluorescently labeled protein. Thus, by utilizing the reporter gene to express the fluorescently labeled protein, previously invisible biometric events are transformed into intuitive, quantitatively detectable optical signals to assist in detection.

[0056] According to an embodiment of the present invention, the genome of the chassis yeast cell further integrates genes encoding the first chimeric receptor and the second chimeric receptor. Thus, genes encoding the first and second chimeric receptors can be stably integrated into the genome of the chassis yeast cell using gene editing technologies such as CRISPR, avoiding problems such as plasmid loss, copy number instability, and antibiotic dependence that may arise from using plasmid expression systems. This ensures long-term, stable, and uniform expression of the chimeric receptor combination on the surface of the engineered yeast. If this method is used to construct engineered yeast, a secretion signal peptide (e.g., the SUC2 signal peptide) can be added before its coding sequence to ensure that the first and second chimeric receptors can be correctly processed, folded, and ultimately transported and anchored to the cell membrane "surface" of the engineered yeast cell to function.

[0057] It should be noted that, in addition to the method of integrating the genes encoding the first chimeric receptor and the second chimeric receptor into the genome of the chassis yeast cell, other methods can be used, such as transient / stable expression systems based on addendum plasmids (e.g., 2μ or CEN plasmids), genome integration at multiple copy sites (e.g., rDNA regions), random integration combined with high-throughput screening, and combinations of different secretory signal peptides (e.g., α-factor) and membrane anchoring methods (e.g., GPI anchoring). As long as the chimeric receptor combination is stably expressed and functions on the surface of the chassis yeast cell of the present invention, it falls within the scope of protection of the present invention.

[0058] According to an embodiment of the present invention, the transmembrane regions of both the first chimeric receptor and the second chimeric receptor are derived from membrane proteins of yeast cells. Therefore, transmembrane regions (YM) derived from the yeast cell itself are selected, such as the yeast Pma1 protein (plasma membrane HM). + The transmembrane domains of ATPase, Suc2 protein (sucrase), Ste2 protein (α factor receptor), Ste3 protein (a factor receptor), or Mid2 protein, etc., are beneficial for maintaining the membrane integrity of engineered yeast cells and improving the expression efficiency and stability of chimeric receptor combinations.

[0059] For example, taking the preferred engineered yeast of the present invention as an example (the target inflammatory factor is IL-1β or IL-17), this engineered strain has extremely high specificity for the target inflammatory factor, producing a strong reporter signal only in the presence of the corresponding ligand, while showing no cross-response to non-target factors (such as TNF-α, IL-6, etc.). At the same time, thanks to the protease / transcription activator protein binary switch and receptor dimerization-dependent activation mechanism, the engineered yeast of the present invention achieves ultra-low background noise in the unstimulated state, while the signal intensity rapidly increases upon activation by a specific ligand, exhibiting an extremely high signal-to-noise ratio. Thus, when using the engineered yeast of the present invention for oral detection, it can accurately and in real time reflect the local inflammatory factor concentration in the complex intestinal microenvironment, and effectively eliminate non-specific interference. It has many advantages such as high sensitivity, high signal-to-noise ratio and extremely low false positive rate, thereby providing a reliable tool for non-invasive dynamic monitoring of inflammatory bowel disease (IBD).

[0060] Reagent test kit This invention provides a kit. According to an embodiment of the invention, the kit comprises: the aforementioned engineered yeast.

[0061] Those skilled in the art will understand that the features and advantages described above for engineered yeast also apply to this kit, and will not be repeated here.

[0062] Pharmaceutical Composition This invention proposes a pharmaceutical composition. According to an embodiment of the invention, the pharmaceutical composition comprises the aforementioned engineered yeast. When the pharmaceutical composition of the embodiment of the invention is administered orally to the intestine, the pharmaceutical composition containing the aforementioned engineered yeast is specifically activated and released only in the pathological microenvironment where the concentration of local inflammatory factors is elevated, thereby achieving lesion-targeted, on-demand drug delivery, further improving the precision and low-side-effect treatment of intestinal diseases (such as inflammatory bowel disease).

[0063] According to embodiments of the present invention, the pharmaceutical composition further comprises pharmaceutically acceptable excipients, such as, but not limited to, one or more solvents, dispersion media, diluents, dispersants, suspending agents, granulation agents, disintegrants, fillers, flow aids, liquid media, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, etc. Excipients include, for example, starch, lactose, or dextrin. Pharmaceutically acceptable excipients are well known in the art. Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugars, and / or combinations thereof.

[0064] In this document, the term "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated; "pharmaceutical acceptable" as used herein specifically means approved by federal regulatory agencies or national governments or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, particularly in humans.

[0065] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent suitable for the specific target dosage form. The use of any conventional excipients, except those incompatible with the pharmaceutical compositions comprising the aforementioned engineered yeast disclosed in this invention, for example, any adverse biological effects produced or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0066] It should be noted that the terms “subject,” “individual,” and “patient” are used interchangeably in this document and refer to a mammal (e.g., a human) being evaluated for treatment and / or being treated. The terms “subject,” “individual,” and “patient” include, but are not limited to, individuals with gastrointestinal diseases, such as individuals with inflammatory bowel disease, such as individuals with ulcerative colitis or Crohn’s disease. Subjects can be humans, but also include other mammals, especially mammals that can be used as laboratory models of human diseases, such as mice and rats.

[0067] It should be noted that the pharmaceutical composition of the present invention is administered orally.

[0068] In this document, the term "treatment" means the use of a drug or product to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in individuals susceptible to disease but not yet diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing symptoms associated with the disease. As used herein, "treatment" encompasses any use of medicine or product to treat, cure, relieve, improve, reduce, or inhibit a disease in an individual, including but not limited to the administration of a pharmaceutical composition described herein to an individual in need.

[0069] use This invention proposes the use of the aforementioned engineered yeast in the preparation of reagents or kits for the detection of inflammatory bowel disease.

[0070] Those skilled in the art will understand that the features and advantages described above for engineered yeast also apply to this application, and will not be repeated here.

[0071] According to an embodiment of the present invention, the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

[0072] This invention proposes the use of the aforementioned engineered yeast in the preparation of reagents or kits for detecting inflammatory factors.

[0073] For example, the inflammatory factors are IL-1β and / or IL-17.

[0074] Those skilled in the art will understand that the features and advantages described above for engineered yeast also apply to this application, and will not be repeated here.

[0075] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0076] Example 1: Construction and functional verification of engineered yeast sensing strains 1. Construction of chassis strains Using wild-type Saccharomyces cerevisiae as the starting strain, an expression fragment containing an inducible promoter driving a green fluorescent protein (GFP) reporter gene was first obtained through plasmid construction. This fragment has homologous sequences (homologous arms) at both ends that are integrated into the yeast genome. Then, this fragment and a CRISPR-Cas9 plasmid targeting the integration site were co-transformed into competent wild-type Saccharomyces cerevisiae cells. After transformation, the cells were plated on selective plates, and single colonies were picked. PCR was used to verify that the GFP reporter gene expression cassette had been correctly integrated into the target genome site. The positive clones verified by PCR were then passaged in non-selective medium to lose the CRISPR plasmid, thus obtaining the chassis yeast strain.

[0077] 2. Construction of IL-1β / IL-17 receptor sensing strains (1) Engineered yeast sensing strain 1 A linear DNA fragment was obtained by PCR amplification, with the structure shown as fragment 1 (targeting IL-1β): Left homologous arm -Ptdh3-SUC2-mIL1R1-YM-TCS-tTA-TTEF1-TFBA1-YM-mIL1R3-SUC2-PTEF 1-Right homologous arm; The functions of each component in the above segment are as follows: Left homologous arm and right homologous arm: respectively attached to the left and right chromosomes at the yeast integration site; Ptdh3 / PTEF1: promoter that drives the expression of chimeric receptor mIL1R1 / R3; SUC2: Signal peptide, secreted protein; mIL1R1: First chimeric receptor (receptor 1), recognizes the inflammatory factor IL1β; YM: Transmembrane domain, which transfers secreted proteins to the yeast cell membrane; TCS: Segmentation and Recognition Sequence; tTA: a transcription activator that drives the expression of inducible promoters in the cell nucleus; TTEF1 / TFBA1: Terminator, terminates chimeric receptor expression; mIL1R3: The second chimeric receptor (receptor 2), which forms a receptor complex with receptor 1; The nucleotide sequence of fragment 1 is shown in SEQ ID NO: 2.

[0078] The linear DNA fragment (fragment 1) and the CRISPR-Cas9 plasmid targeting the yeast genome integration site in step 1 were co-transformed into competent cells of the chassis yeast strain obtained in step 1. After transformation, the cells were plated on selective plates and cultured for 3-4 days. Single colonies were picked, genomic DNA was extracted, and colony PCR was performed to verify that the DNA fragments had been correctly integrated into the strain genome. The positive clone strain that passed the PCR verification was named engineered yeast sensing strain 1, which is able to sense the IL-1β inflammatory factor.

[0079] (2) Engineered yeast sensing strain 2 A linear DNA fragment was obtained by PCR amplification, and its structure is shown as fragment 2 (targeting IL-17): Left homology arm-Ptdh3-SUC2-mIL17RA-YM-TCS-tTA-TTEF1-TFBA1-YM-mIL17RC-SUC2-PT EF1 - Right homologous arm.

[0080] The functions of each component in the above segment are as follows: Left homologous arm and right homologous arm: respectively attached to the left and right chromosomes at the yeast integration site; Ptdh3 / PTEF1: promoter that drives the expression of chimeric receptor mIL1R1 / R3; SUC2: Signal peptide, secreted protein; mIL17RA: First chimeric receptor (receptor 1), recognizes the inflammatory cytokine IL17; YM: Transmembrane domain, which transfers secreted proteins to the yeast cell membrane; TCS: Segmentation and Recognition Sequence; tTA: a transcription activator that drives the expression of inducible promoters in the cell nucleus; TTEF1 / TFBA1: Terminator, terminates chimeric receptor expression; mIL17RC: The second chimeric receptor (receptor 2) recognizes the IL17 factor and forms a receptor complex with receptor 1; The nucleotide sequence of fragment 2 is shown in SEQ ID NO: 3.

[0081] The linear DNA fragment (fragment 2) and the CRISPR-Cas9 plasmid targeting the yeast genome integration site in step 1 were co-transformed into competent cells of the chassis yeast strain obtained in step 1. After transformation, the cells were plated on selective plates and cultured for 3-4 days. Single colonies were picked, genomic DNA was extracted, and colony PCR was performed to verify that the DNA fragments had been correctly integrated into the strain genome. The positive clone strain that passed the PCR verification was named engineered yeast sensing strain 2, which can sense the IL-17 inflammatory factor.

[0082] 3. Validation of the sensitivity and specificity of engineered yeast sensing strains (1) Engineered yeast sensing strain 1 Sensitivity tests were performed on engineered yeast sensing strain 1 and chassis yeast strain. The specific steps are as follows: Control group 1: Yeast strains were cultured in YPD medium in a chassis. Control group 2: Yeast strains were cultured in YPD medium with an additional 10 ng / mL IL-1β inducing factor. Control group 3: Engineered yeast sensing strain 1 was cultured in YPD medium; Experimental group: Engineered yeast sensing strain 1 was cultured in YPD medium and an additional 10 ng / mL IL-1β inducing factor was added.

[0083] Each group of bacterial strains was cultured in a shaker at 30℃ and 220 rpm. Samples were taken 10 h after culture, the bacterial cells were collected by centrifugation, washed twice with PBS and resuspended, and the GFP fluorescence intensity was detected by flow cytometry.

[0084] The sensitivity test results of engineered yeast sensing strain 1 are shown in the figure. Figure 1 .

[0085] The results showed that the GFP signal of the chassis yeast strain did not change significantly in either the presence or absence of IL-1β stimulation (see details). Figure 1 (A and B in the text); while the engineered yeast sensing strain 1 maintained a low background fluorescence of 10.8% under unstimulated conditions (see details). Figure 1 (C in the text), and after stimulation with IL-1β-inducible factor, the GFP signal significantly increased to 14.9% (see details). Figure 1 The D in the figure indicates that the engineered yeast sensing strain 1 has a high sensitivity response to IL-1β.

[0086] (2) Engineered yeast sensing strain 2 Sensitivity tests were performed on engineered yeast sensing strain 2 and chassis yeast strain. The specific steps are as follows: Control group 1: Yeast strains were cultured in YPD medium in a chassis. Control group 2: Yeast strains were cultured in YPD medium and supplemented with 10 ng / mL IL-17 inducing factor. Control group 3: Engineered yeast sensing strain 2 was cultured in YPD medium; Experimental group: Engineered yeast sensing strain 2 was cultured in YPD medium and an additional 10 ng / mL IL-17 inducing factor was added.

[0087] Each group of bacterial strains was cultured in a shaker at 30℃ and 220 rpm. Samples were taken 10 h after culture, the bacterial cells were collected by centrifugation, washed twice with PBS and resuspended, and the GFP fluorescence intensity was detected by flow cytometry.

[0088] The sensitivity test results of engineered yeast sensing strain 2 are shown in the figure. Figure 2 .

[0089] The results showed that the GFP signal of the chassis yeast strain did not change significantly in either the presence or absence of IL-17 stimulation (see details). Figure 2 (A and B in the text); while the engineered yeast sensing strain 2 maintained a low background fluorescence of 10.2% under unstimulated conditions (see details). Figure 1 The GFP signal increased significantly to 12.8% after stimulation with IL-17-inducible factor, indicating that the engineered yeast sensing strain 2 has a high sensitivity response to IL-17.

[0090] The above results demonstrate that engineered yeast strain 1, capable of sensing IL-1β, and engineered yeast strain 2, capable of sensing IL-17, were successfully constructed. Furthermore, under in vitro conditions, they exhibited high sensitivity (significant activation at 10 ng / mL) and high specificity (no cross-reactivity) for their homologous inflammatory factors (IL-1β or IL-17), respectively, meeting the basic requirements for a non-invasive IBD monitoring platform.

[0091] Example 2: Assessment of the diagnostic potential of engineered yeast sensing strains for IBD in animal disease models The engineered yeast sensing strains 1 and 2 obtained in Example 1 were used to evaluate the potential for differential diagnosis of IBD in animal disease models. The specific steps are as follows: Six-week-old male C57BL / 6 mice were randomly divided into four groups (n=3): control group 1 (NC-engineered yeast sensing strain 1), control group 2 (NC-engineered yeast sensing strain 2), experimental group 1 (DSS-engineered yeast sensing strain 1), and experimental group 2 (DSS-engineered yeast sensing strain 2). The treatments for each group of mice were as follows: Control group 1: Drink normal water and receive gavage with engineered yeast sensing strain 1 (2×10⁶) on days 5, 6, and 7. 8 (units / day) Control group 2: Drink normal water and receive gavage with engineered yeast sensing strain 2 (2×10⁶) on days 5, 6, and 7. 8 (units / day) Experimental Group 1: Students were allowed free access to water containing 2.5% (w / v) sodium dextran sulfate (DSS) for 7 consecutive days to induce an acute colitis model (simulating the active phase of IBD). On days 5, 6, and 7 of DSS induction (i.e., the peak of IBD disease activity), they were administered engineered yeast sensing strain 1 (2 × 10⁻⁶) by gavage. 8 (units / day) Experimental Group 2: Students were allowed free access to water containing 2.5% (w / v) sodium dextran sulfate (DSS) for 7 consecutive days to induce an acute colitis model (simulating the active phase of IBD). On days 5, 6, and 7 of DSS induction (i.e., the peak of IBD disease activity), they were administered engineered yeast sensing strain 2 (2 × 10⁻⁶) by gavage. 8 (units / day).

[0092] Fresh fecal samples were collected from each group of mice 3 hours after each gavage. The samples were homogenized with PBS, filtered, and then the GFP fluorescence intensity level of yeast cells was detected by flow cytometry.

[0093] The results of GFP fluorescence intensity detection in yeast cells in each group of fecal samples are shown below. Figure 3 .

[0094] The results showed that the fluorescence intensity of the feces of mice in experimental group 1 was higher than that of healthy mice in control group 1, and the fluorescence intensity of the feces of mice in experimental group 2 was higher than that of healthy mice in control group 2.

[0095] The above results demonstrate that the yeast sensing strains 1 and 2 constructed in this invention can specifically respond to IBD-related local inflammatory signals (IL-1β and IL-17) in the complex microenvironment of the living gut and convert the inflammatory signals into quantifiable fluorescent signals.

[0096] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0097] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An engineered yeast, characterized in that, The engineered yeast includes: Chassis yeast cells have a genome that integrates reporter gene expression units regulated by transcriptional activating proteins. and the chimeric receptor ensemble expressed on the surface of the yeast cells in the chassis; The chimeric receptor combination includes at least one set of chimeric receptor pairs, and each set of chimeric receptor pairs includes a first chimeric receptor and a second chimeric receptor; Both the first chimeric receptor and the second chimeric receptor contain an extracellular region, a transmembrane region, and an intracellular region, with the transmembrane region connected to the extracellular region and the intracellular region connected to the transmembrane region. The extracellular region of the first chimeric receptor can specifically bind to inflammatory factors; The intracellular region of the first chimeric receptor contains a protease catalytic domain; The extracellular region of the second chimeric receptor can specifically recognize and bind to the complex formed by the inflammatory factor and the extracellular region of the first chimeric receptor, thereby causing the first chimeric receptor and the second chimeric receptor to dimerize. The intracellular region of the second chimeric receptor includes the cleavage recognition sequence of the protease and the transcriptional activation protein attached to its C-terminus; The dimerization causes the protease catalytic domain to cleave the cleavage recognition sequence, releasing the transcriptional activator protein into the cell nucleus, thereby activating the reporter gene expression unit.

2. The engineered yeast according to claim 1, characterized in that, The transcriptional activating proteins include one or more of rtTA, GAL4, Cat8, Sip4, Aca1, and Aca2; Optionally, the transcriptional activating protein is rtTA, and the reporter gene expression unit contains a tetracycline-responsive element.

3. The engineered yeast according to claim 1, characterized in that, The inflammatory factor is IL-1β or IL-17; Optionally, the protease catalytic domain includes one or more of the following: tobacco etch virus protease catalytic domain, SUMO protease catalytic domain, Atg4p protease catalytic domain, HRV 3C protease catalytic domain, and NEDP1 protease catalytic domain. Optionally, the protease catalytic domain is a tobacco etch virus protease catalytic domain; Optionally, the protease catalytic domain is the tobacco etch virus protease catalytic domain, and the amino acid sequence of the cleavage recognition sequence is shown in SEQ ID NO:

1.

4. The engineered yeast according to claim 1, characterized in that, The extracellular region of the first chimeric receptor is the extracellular domain of the IL-1R1 protein, and the extracellular region of the second chimeric receptor is the extracellular domain of the IL-1RAcP protein. Optionally, the extracellular region of the first chimeric receptor is the extracellular domain of the IL-17RA protein, and the extracellular region of the second chimeric receptor is the extracellular domain of the IL-17RC protein.

5. The engineered yeast according to claim 1, characterized in that, The reporter gene expression unit also includes the coding sequence of a fluorescently labeled protein; Optionally, the genome of the chassis yeast cell also integrates genes encoding the first chimeric receptor and the second chimeric receptor; Optionally, the transmembrane regions of both the first chimeric receptor and the second chimeric receptor are derived from membrane proteins of yeast cells.

6. A reagent kit, characterized in that, The kit includes: The engineered yeast according to any one of claims 1 to 5.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: The engineered yeast according to any one of claims 1 to 5.

8. Use of the engineered yeast according to any one of claims 1 to 5 in the preparation of reagents or kits for the detection of inflammatory bowel disease.

9. The use according to claim 8, characterized in that, The inflammatory bowel disease mentioned refers to ulcerative colitis or Crohn's disease.

10. Use of the engineered yeast according to any one of claims 1 to 5 in the preparation of reagents or kits for detecting inflammatory factors.

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