Bile salt Bactosensor and its use for diagnostic and therapeutic purposes

Abstract

We designed a synthetic bile salt receptor using TcpP as a sensing domain connected to the CadC system of Escherichia coli, which activates transcription upon dimerization. The performance of the system was assayed against various selected promoters, demonstrating the fine-tunable response that can be achieved by varying the expression level of the bile salt receptor. By performing multiple rounds of guided evolution of the TcpP sensor, we obtained a panel of mutants with lower detection limits and greater sensitivity. Finally, we demonstrate that this Bacto sensor can detect pathological bile salt concentrations in samples from patients with liver dysfunction. Thus, the present invention relates to a bile salt Bacto sensor and its use for diagnostic and therapeutic purposes.

Description

[Technical Field]

[0001] This invention relates to medicine, particularly the fields of synthetic biology and hepatology. [Background technology]

[0002] The liver is a vital organ that coordinates metabolic, detoxification, and immunological processes. Liver diseases, including hepatitis, cirrhosis, fatty liver disease, and cancer, are major public health issues and require large-scale screening methods for prevention, diagnosis, and therapeutic monitoring. Liver biopsy and ultrasound-based elastography are the most common methods for diagnosing and monitoring the progression of liver disease. However, these techniques remain limited due to the need for high-performance equipment and skilled technicians. Liver function can also be monitored by quantifying serum enzyme activity and bilirubin, but these markers are not entirely specific and are only detectable when damage has already progressed. Liver function is usually monitored by quantifying multiple enzyme activities together, because they are not specific to each other. Serum and urine bile salts are another biomarker for the early diagnosis of liver dysfunction, but current detection methods are impractical and difficult to measure.

[0003] Patent Document 1 discloses a bile salt sensor, particularly a bile salt transcription sensor in Bacteroides thetaiotaomicron that uses bile sensor proteins such as BreR and VFA0359 from Vibrio fischeri. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] International Publication No. 2018 / 049362 [Overview of the project]

[0005] The present invention is defined by the claims. More specifically, the present invention relates to a bile salt bactosensor and its use for diagnostic and therapeutic purposes. [Brief explanation of the drawing]

[0006] [Figure 1] Figure 1 illustrates the rewiring of pathogenicity sensing to a modularized synthetic receptor platform. It shows a schematic diagram of isolating the transmembrane and periplasmic domains of TcpP and TcpH from the pathogen V. cholerae, which constitute the bile salt sensing module, and rewiring this module to a modularized E. coli synthetic receptor platform. The TcpP sensing domain, which detects bile salts, is attached to a synthetic receptor that activates GFP expression in the presence of bile salts. [Figure 2] Figure 2 shows the system performance of a constitutively expressed CadC-TcpP_TcpH synthesis receptor platform. (A) Shows the architecture of the expression system encoding CadC-TcpP and TcpH controlled by homeostatic promoters (P14, P10, P9 for TcpP, and P5 for TcpH). (B) Shows the response of various constructs to increased concentrations of bile salt (taurocholic acid). Regions indicated by (Δ) show serum bile salt concentrations in healthy humans (4.8±0.6 μM, Δ), patients with chronic liver disease (51.3±3.8 μM, ΔΔ), and patients with drug-induced liver injury (110±3.4 μM, ΔΔΔ). (C) Activation factor of CadC-TcpP controlled by P14, P10, and P9 is measured in the presence of 120 μM taurocholic acid. [Figure 3] Figure 3 shows specificity testing of the rewired TcpP-Cadc system with various bile salts. [Figure 4] Figure 4 shows the sensitivity manipulation of the TcpP bile salt sensing domain of selected TcpP functional mutants in the taurocholic acid titration curve. [Figure 5A-B]Figures 5A-B show the response of bile salt bactosensors in clinical samples. (A) Shows the response of bacterial cells containing the P9-CadC-TcpP_P5-TcpH synthesis receptor in serum clinical samples. (B) Shows the response of bacterial cells containing the P9-CadC-TcpP_P5-TcpH synthesis receptor in urine clinical samples. [Figure 6] Figure 6 shows a colorimetric assay for Bactosensor-mediated bile salt detection. It presents bile salt specificity profiles of SV_3-3-18 mutants of TcpP, characterized using an SV_3-3-18-LacZ sensor. The SV_3-3-18-LacZ response was quantified as ΔA580 (difference in absorbance at 580 nm (A580) with and without the ligand bile salt). The bar graph and error correspond to the mean values ​​of three experiments performed in triplicate mode. Prior to flow cytometry analysis, cells growing in the logarithmic phase were incubated with bile salt for 4 hours. [Figure 7] Figure 7 shows the detection of pathological bile salts in clinical samples using a bactosensor. Analysis results for 21 serum clinical samples were compared between TcpP18LacZ and the bile salt assay kit. The response of TcpP to SV_3-3-18-LacZ is shown in black bars on the left axis. The total bile salt concentration in serum, measured by the bile salt enzyme assay kit, is shown in green asterisks on the right axis. [Modes for carrying out the invention]

[0007] (definition) As used herein, the term “amino acid residue” is used to include any natural or synthetic amino acid residue, and is primarily used to refer to an amino acid residue selected from the group consisting of 20 naturally occurring amino acids, namely, alanine (Ala or A), cysteine ​​(Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues.

[0008] As used herein, the terms “polypeptide,” “peptide,” and “protein” are interchangeable to refer to polymers of amino acids of any length. These terms also encompass modified amino acid polymers, such as those modified by disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. When described in the context of gene therapy, a polypeptide refers to any fragment or genetically modified derivative thereof that retains the desired biochemical function of the respective intact polypeptide or intact protein.

[0009] As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may include modified nucleotides, such as methylated nucleotides, and nucleotide analogs, which may be interposed by non-nucleotide components. Modification of the nucleotide structure, if present, may be made before or after polymer formation. As used herein, the term polynucleotide refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein, which is a polynucleotide, encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to constitute the double-stranded form.

[0010] As used herein, the term “identity” refers to the exact correspondence between nucleotides and nucleotides or amino acids in two polynucleotide or polypeptide sequences. The percentage of identity can be determined by directly comparing the sequence information of two molecules by aligning the sequences, counting the number of exact matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. In this invention, a first amino acid sequence having at least 90% identity with a second amino acid sequence means that the first sequence has 90;91;92;93;94;95;96;97;98;99; or 100% identity with the second amino acid sequence. Sequence identity is often determined as a percentage of identity (or similarity or homology), with a higher percentage indicating greater similarity between the two sequences. For sequence alignment methods for comparison, various programs and alignment algorithms well known in the art are used, including: Smith and Waterman, Adv. Appl. Math., 2:482, 1981; Needleman and Wunsch, J. Mol. Biol., 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444, 1988; Higgins and Sharp, Gene, 73:237-244, 1988; Higgins and Sharp, CABIOS, 5:151-153, 1989; Corpet et al., Nuc. Acids Res., 16:10881-10890, 1988; Huang et al., Comp. Appls This is described in Biosci., 8:155-165, 1992; and Pearson et al., Meth.Mol.Biol., 24:307-31, 1994). Altschul et al., Nat.Genet., 6:119-129, 1994, provides a detailed discussion of sequence alignment methods and homology calculations.As an example, sequence comparison can be performed using an alignment tool such as ALIGN (Myers and Miller, CABIOS 4:11-17, 1989) or LFASTA (Pearson and Lipman, 1988) (Internet Program (trademark), 1996, W.R. Pearson and the University of Virginia, fasta20u63 version 2.0u63, published in December 1996). ALIGN compares the entire sequences with each other, and LFASTA compares regions of local similarity. These alignment tools and their respective tutorials are available, for example, on the NCSA website on the Internet. Alternatively, for comparison of amino acid sequences of more than about 30 amino acids, the Blast2 sequences function can be used with the default BLOSUM62 matrix set to default parameters (gap existence cost 11, gap cost per residue 1). When aligning short peptides (less than about 30 amino acids), it is necessary to use the Blast2 sequences function with the PAM30 matrix set to default parameters (starting gap penalty 9, extension gap penalty 1). The BLAST sequence comparison system is available, for example, from the NCBI website, and is also referred to in Altschul et al., J. Mol. Biol., 215:403-410, 1990; Gish and States, Nature Genet., 3:266-272, 1993; Madden et al., Meth. Enzymol., 266:131-141, 1996; Altschul et al., Nucleic Acids Res., 25:3389-3402, 199,7; and Zhang and Madden, Genome Res., 7:649-656, 1997.

[0011] As used herein, the expression "derived from" refers to the process used to isolate, derive, or produce a second component (e.g., a second polypeptide different from the first polypeptide) from a first component (e.g., a first polypeptide) or information of the first component.”

[0012] As used herein, the term "fusion protein" refers to a single polypeptide chain having at least two polypeptide domains that are not normally present in a single native polypeptide. Thus, a protein of natural origin is not a "fusion protein" as used herein. Preferably, a polypeptide of interest (e.g., a TcpP polypeptide) is fused via a peptide bond to at least one heterologous polypeptide (e.g., a DNA binding domain), and the fusion protein can also include a linking region of amino acids between amino acid portions derived from separate proteins.”

[0013] As used herein, the term "heterologous polypeptide" refers to a polypeptide that is not derived from the same protein to which the above-mentioned heterologous polypeptide is fused.”

[0014] As used herein, the term "linker" refers to the sequence of at least one amino acid that links a polypeptide of interest to a heterologous polypeptide in a fusion protein. Such linkers can be useful for preventing steric hindrance. Typically, a linker contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 70 amino acids.”

[0015] As used herein, the term "TcpP" refers to the toxin-coregulating ciliary biosynthesis protein P of Vibrio cholerae. TcpP is a transmembrane transcription factor, and it has been shown that a series of bile salts induce intermolecular disulfide bonds in the periplasmic domain, resulting in the dimerization of the transmembrane transcription factor TcpP. In particular, the TcpP protein contains a transmembrane domain and a periplasmic sensing domain. An example amino acid sequence of TcpP is shown as Sequence ID No. 1. Sequence ID 1 >sp|P29485|TCPP_VIBCH Toxin coregulated pilus biosynthesis protein P OS=Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961) OX=243277 GN=tcpP PE=4 SV=2, the transmembrane domain is shown in parentheses, square brackets, and curly brackets. The periplasm sensing domain is shown in parentheses and angle brackets. MGYVRVIYQFPDNLWWNECTNQVYYAQDPMKPERLIGTPSIIQTKLLKILCEYHPAPCPNDQIIKALWPHGFISSESLTQAIKRTRDFLNDEHKTLIENVKLQGYRINIIQVIVSENVVDEADCSQKKSVKERIKIEWGKIN([〔VVPYLVFSALYVALLPVIWWS〕] <ygqwyqhelagithdlrdlarlpgitiqklseqkltfaidqhqcsvnyeqktlectkn>)

[0016] As used herein, the term "TcpH" refers to the toxin-coregulating ciliary biosynthesis protein H of Vibrio cholera. An example amino acid sequence of TcpH is shown as Sequence ID No. 2. SEQ ID NO:2 >sp|P29489|TCPH_VIBCH Toxin coregulated pilus biosynthesis protein H OS=Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961) OX=243277 GN=TcpH PE=4 SV=2 MHKKLKAWGGATGLFVVALGVTIIALPMRQKNSHGTMIIDGTVTQIFSTYQGNLSNVWLTQTDPQGNVVKSWTTRYQTLPDPSSQKLNLIPDYSQSNASRDYNVLSIYQLGKGCFLAFPYKLLTAEKMWFSCQSDF

[0017] As used herein, “DNA-binding domain” refers to, but is not limited to, a motif capable of binding to a specific DNA sequence (e.g., a genomic DNA sequence). A DNA-binding domain has at least one motif that recognizes and binds to single-stranded or double-stranded DNA. A DNA-binding domain can interact with DNA in a sequence-specific or non-sequence-specific manner.

[0018] As used herein, the term “CadC transcription activator” has its general meaning in this technology and refers to CadC, a membrane insertion transcription regulator of Escherichia coli. CadC, with the simultaneous availability of lysine, activates the expression of the cadBA operon at low external pH, thereby adapting to moderate acidic stress. CadC is a representative ToxR-like protein that combines sensory signaling and DNA-binding activity within a single polypeptide. In particular, CadC consists of a C-terminal periplasmic pH-sensing domain, a single transmembrane helix, and an N-terminal cytoplasmic winged helix-turn-helix DNA-binding domain (Buchner S, Schlundt A, Lassak J, Sattler M, Jung K. Structural and Functional Analysis of the Signal-Transducing Linker in the pH-Responsive One-Component System CadC of Escherichia coli. J Mol Biol. 2015 Jul 31;427(15):2548-61). CadC dimerizes via its C-terminal periplasmic pH-sensing domain. Therefore, the expression "CadC transcription activator DNA-binding domain of E. coli" refers to the cytoplasmic domain of CadC, which can regain its function through oligomerization of its C-terminal fusion domain.

[0019] As used herein, the term “recombinant” refers to an artificial combination of two distinct sequence segments, for example, by manipulating isolated segments of amino acids or nucleic acids by chemical synthesis or genetic engineering techniques.

[0020] As used herein, the term “expression cassette” refers, where appropriate, to a nucleic acid sequence capable of promoting the expression of a polynucleotide encoding a target polypeptide incorporated into the expression cassette described above. When introduced into a host cell, the expression cassette can induce cellular mechanisms, in particular, to transcribe the incorporated polynucleotide encoding the target polypeptide into RNA, which is then typically further processed and finally translated into the target polypeptide. The expression cassette can be contained within an expression vector, as will be described in more detail below. The individual elements of the expression cassette in this invention will be described in detail later.

[0021] As used herein, the term “promoter” refers to a nucleic acid sequence that promotes the transcription of a polynucleotide of interest. Promoters are operably ligated to the polynucleotide of interest. Promoters may also form part of a promoter / enhancer component. While the physical boundary between “promoter” and “enhancer” components is not always clear, the term “promoter” typically refers to the site on a nucleic acid molecule to which RNA polymerase and / or any associated factors bind and initiate transcription. Enhancers transiently and spatially enhance promoter activity. Many promoters with transcriptional activity across a wide range of cell types are known in the prior art.

[0022] As used herein, the term “operatably linked” refers to linking two polynucleotides, in particular, between an expression regulatory sequence (e.g., a promoter) and the polynucleotide of interest.

[0023] As used herein, the term “vector” refers to a substance capable of introducing a nucleic acid sequence into a target cell (e.g., non-viral vectors, particulate carriers, and liposomes). Typically, “vector construct,” “expression vector,” and “gene transfer vector” mean any nucleic acid construct capable of inducing the expression of a desired nucleic acid and introducing a nucleic acid sequence into a target cell. Thus, the terms above include cloning and expression media, as well as viral vectors.

[0024] As used herein, the term “host cell” may refer to any of the many cells commonly used in the production of exogenous polypeptides or proteins, including prokaryotic host cells.

[0025] As used herein, the term “probiotics” refers to viable microorganisms that, when taken in sufficient quantities, exert positive effects on health, wellness, and wellness beyond their conventional nutritional benefits. Probiotic microorganisms are defined as “viable microorganisms that, when administered in appropriate amounts, provide health benefits to a host” (FAO / WHO, 2001).

[0026] As used herein, the term “gene transfer” refers to a wide variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, and DEAE-dextran gene transfer. Gene transfer with the vector of the present invention can be performed into host cells by any conventional means known to those skilled in the art. For example, the gene transfer may be transient.

[0027] As used herein, the term “bile salt” has its general meaning in this art and is synthesized in the liver from cholesterol, conjugates with glycine or taurine, and is secreted into the bile along with cholesterol and lecithin. Examples of bile salts include dihydroxycholic acid, e.g., deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, and taurochenodeoxycholic acid, and trihydroxycholic acid, e.g., salts of cholic acid, glycocholic acid, and taurocholic acid. Alkali salts include sodium and potassium. In some embodiments, the bile salt is a primary bile salt. Examples of primary bile salts include dihydroxycholic acid, e.g., chenodeoxycholic acid, glycochenodeoxycholic acid, and taurochenodeoxycholic acid, and trihydroxycholic acid, e.g., salts of cholic acid, glycocholic acid, and taurocholic acid.

[0028] As used herein, the term “output molecule” refers to a polynucleotide or polypeptide expressed in response to a specific signal, such as the presence of a bile salt.

[0029] As used herein, the term “therapeutic polypeptide” refers to any type of protein or polypeptide that exerts a therapeutic effect on a subject. The term “therapeutic polynucleotide” refers to any type of polynucleotide that exerts a therapeutic effect on a subject.

[0030] As used herein, the term “Subject” refers to any mammalian living organism as used herein. The term “Subject” includes, but is not limited to, humans, non-human primates, e.g., chimpanzees and other apes and monkey species, domesticated animals, e.g., cattle, sheep, pigs, goats, and horses, domesticated mammals, e.g., dogs and cats, and laboratory animals, including rodents, e.g., mice, rats, and guinea pigs. The terms above are not intended to refer to a specific age or sex. Therefore, adult and neonatal subjects, as well as fetuses, whether male or female, are intended to be included in its scope.

[0031] As used herein, the term “sample” refers to any volume of liquid or suspension in which the bile salt to be measured may be present in the solution.

[0032] As used herein, the terms “liver dysfunction” or “hepatic dysfunction” refer to a state in which liver function is reduced compared to a normal state. Hepatic dysfunction is characteristic of liver disease.

[0033] As used herein, the term “non-alcoholic fatty liver disease” has its general meaning in this art and is used to refer to a spectrum of disorders resulting from the accumulation of fat in liver cells in individuals with no history of extremely heavy alcohol consumption. NAFLD refers to fatty liver in its mildest form. The term NAFLD is also used to encompass more severe and advanced forms of non-alcoholic steatohepatitis (NASH), cirrhosis, hepatocellular carcinoma, and virus-induced (e.g., HIV, hepatitis) fatty liver disease.

[0034] As used herein, the terms “drug-induced liver disease” or “toxic liver injury” are used to describe cases in which an active agent causes damage to the liver.

[0035] As used herein, the terms “alcoholic liver disease” or “alcoholic liver injury” refer to diseases resulting from the accumulation of fat in liver cells, at least in part, caused by alcohol consumption. Examples include, but are not limited to, alcoholic simple fatty liver, alcoholic steatohepatitis (ASH), alcoholic liver fibrosis, and alcoholic cirrhosis. It should be noted that alcoholic steatohepatitis is also called alcoholic fatty hepatitis and includes alcoholic liver fibrosis.

[0036] As used herein, the term “risk” in the context of the present invention may mean either the “absolute” risk or the “relative” risk of an event in question with respect to the probability that an event will occur over a particular period of time. Absolute risk can be measured either against a post-hoc measurement of actual observations in the relevant period cohort, or against an index value derived from a statistically valid historical cohort tracked over the relevant period. Relative risk refers to the ratio of the absolute risk of an event in question to either the absolute risk of a low-risk cohort or the mean population risk, and may vary depending on how the clinical risk factor is assessed. The odds ratio, which is the ratio of positive events to negative events in a given test outcome, is also commonly used for non-conversion (the odds follow the formula p / (1-p), where p is the probability of the event and (1-p) is the probability of no event). “Risk assessment” or “risk evaluation” in the context of the present invention includes predicting the probability, odds, or likelihood that an event or disease state may occur, or the incidence of conversion from one event or disease state to another. Furthermore, risk assessment may include predictions of future clinical parameters, conventional laboratory risk factor values, or other indicators of recurrence, in an absolute or relative sense, relative to the population measured to date. The methods of the present invention may be used to perform sequential or categorical measurements of conversion risk to diagnose and define the risk spectrum of a category of subjects designated as at risk of conversion. In the categorical case, the present invention can be used to distinguish between a normal subject cohort and other subject cohorts at higher risk. In some embodiments, the present invention may be used to distinguish between those that are normal and those that are at risk.

[0037] As used herein, the term “liver transplant” has a general meaning in this art and includes partial or whole liver transplants in which a donor’s liver is partially or completely resected and transplanted into a recipient. Partial liver transplants are classified into orthotopic partial liver transplants and ectopic partial liver transplants, etc., depending on the surgical method, and the present invention can be applied to any of them. In a partial liver transplant, a donor liver graft or partial liver graft, corresponding to about 30-50% of the normal liver volume in the recipient, is typically transplanted as a graft into a recipient whose liver has been completely resected.

[0038] As used herein, the term “graft rejection” is defined as functional and structural deterioration of an organ due to an active immune response expressed by the recipient, and is distinct from non-immunological causes of organ dysfunction. Graft rejection can be acute or chronic. The term “acute rejection” as used herein refers to rejection of the transplanted organ that develops within the first 5 to 60 days after transplantation. This is generally a sign of cell-mediated immune injury. Both delayed-type hypersensitivity and cytotoxic mechanisms are thought to be involved. The immune injury targets HLA, and possibly other cell-specific antigens expressed by tubular epithelium and vascular endothelium. The term “chronic rejection” as used herein refers to rejection of the transplanted organ that develops within the first 30 to 120 days after transplantation. Furthermore, the term “chronic rejection” refers to the result of a combination of immunological injury (e.g., chronic rejection) and non-immunological injury (e.g., hypertensive nephrosclerosis or nephrotoxicity of immunosuppressants such as cyclosporine A) that occurs months or years after transplantation and ultimately leads to allograft fibrosis and sclerosis associated with progressive renal loss.

[0039] As used herein, the terms “treatment” or “to treat” include both preventive or deterrent treatments and curative or disease-modifying treatments, including treatments for patients at risk of developing or suspected of developing a disease, and for patients diagnosed with a disease or medical condition, including treatments for patients who are diseased or have a disease or medical condition, and including treatments for An induction regimen may use a “loading regimen” (partially or entirely), which may include administering a higher dose of the drug than the physician may use in the maintenance regimen, administering the drug more frequently than the physician may use in the maintenance regimen, or both. The terms “maintenance regimen” or “maintenance period” refer to a treatment regimen (or part of a treatment regimen) used to maintain a patient at the time of treatment of a disease, for example, to keep the patient in remission for a long period (several months or several years). A maintenance regimen may use continuous treatment (e.g., administering the drug at regular intervals, e.g., weekly, monthly, yearly) or intermittent treatment (e.g., treatment with interruption, intermittent treatment, treatment in relapse, or treatment upon meeting certain conditions [e.g., pain, signs of the disease]).

[0040] As used herein, the term “effective dose” refers to a sufficient amount of prokaryotic host cells to obtain a beneficial effect.

[0041] As used herein, the term “biosensor device” has its general meaning in this technology and refers to a device that measures or detects a target by converting the interaction between a sensor and a recognition molecule into a signal such as an electrical signal.

[0042] As used herein, the term “endonuclease” refers to an enzyme that cleaves phosphodiester bonds within polynucleotide chains. Some, such as deoxyribonuclease I, cleave DNA relatively nonspecifically (regardless of sequence), while many, typically called restriction endonucleases or restriction enzymes, cleave only at highly specific nucleotide sequences. The mechanism behind endonuclease-based genome inactivation generally requires a first step of single- or double-strand cleavage of DNA, which can then trigger two distinct cellular mechanisms for DNA repair (error-prone non-homologous end joining (NHEJ) and high-fidelity homologous recombination repair (HDR)), which can be utilized for DNA inactivation. DNA-targeted endonucleases can be naturally occurring endonucleases (e.g., bacterial meganucleases) or can be artificially created (e.g., particularly engineered meganucleases, TALENs, or ZFNs). As used herein, the term "TALEN" has its general meaning in this technology and refers to a transcription activator-like effector nuclease, which is an artificial nuclease that can be used for editing target genes. As used herein, the term "ZFN" or "zinc finger nuclease" has its general meaning in this technology and refers to a zinc finger nuclease, which is an artificial nuclease that can be used for editing target genes. As used herein, the term "CRISPR-related endonuclease" has its general meaning in this technology and refers to a segment of prokaryotic DNA containing short repeating base sequences, which are associated clustered and regularly arranged short palindromic repeat sequences.

[0043] As used herein, the term “food” means a liquid (i.e., beverage), solid, or semi-solid dietary composition, in particular a complete food composition (alternative food) that does not require additional nutritional intake or food supplement composition. As used herein, the term “food component” or “feed component” includes preparations that are added to or may be added to functional foods or functional ingredients as nutritional supplements. “Nutritional food” or “nutritional supplement” food or “functional” food means an ingredient that contains components that have beneficial effects on health or components that can improve physiological functions. “Food supplement” means an ingredient intended to complement a normal diet.

[0044] (polypeptide) The first object of the present invention relates to a bile salt sensing domain having the amino acid sequence shown in Sequence ID No. 3, - X 47 This represents N, D, W, Y, T, V, or F. - X 48 This indicates Y or F. - X 49 This represents E, G, V, I, L, S, or K. - X 50 represents Q, V, H, A, T, D, L, or S. Sequence ID 3: bile salt domain YGQWYQHELAGITHDLRDLARLPGITIQKLSEQKLTFAIDQHQCSV-X 47 -X 48 -X 49 -X 50 -KTLECTKN

[0045] In some embodiments, the bile salt domain includes the amino acid sequences shown in SEQ ID NOs. 25-33, disclosed in Table A.

[0046] In some embodiments, the bile salt domain does not consist of the amino acid sequence shown in SEQ ID NO: 24. [Table 1]

[0047] A further object of the present invention relates to a TcpP polypeptide having the sequence shown in SEQ ID NO: 34, - X 47 represents N, D, W, Y, T, V, or F, - X 48 represents Y or F, - X 49 represents E, G, V, I, L, S, or K, - X 50 represents Q, V, H, A, T, D, L, or S. SEQ ID NO: 34 VVPYLVFSALYVALLPVIWWS-YGQWYQHELAGITHDLRDLARLPGITIQKLSEQKLTFAIDQHQCSV-X 47 -X 48 -X 49 -X 50 -KTLECTKN

[0048] In some embodiments, the TcpP polypeptide comprises the amino acid sequences shown in SEQ ID NOs: 36 to 44 disclosed in Table B.

[0049] In some embodiments, the TcpP polypeptide does not consist of the amino acid sequence shown in SEQ ID NO: 35 disclosed in Table B. [Table 2]

[0050] A further object of the present invention relates to a fusion protein in which the TcpP polypeptide of the present invention is fused with a heterologous polypeptide.

[0051] In some embodiments, the heterologous polypeptide can be fused to the N-terminus or C-terminus of the TcpP polypeptide of the present invention.

[0052] In some embodiments, the TcpP polypeptide of the present invention is fused to the heterologous polypeptide either directly or via a linker.

[0053] In some embodiments, the heterologous polypeptide is a DNA-binding domain.

[0054] In some embodiments, the heterologous polypeptide is the CadC transcription activator DNA-binding domain of E. coli. In some embodiments, the CadC transcription activator DNA-binding domain of E. coli contains an amino acid sequence having at least 90% identity with SEQ ID NO: 45. Sequence ID 45 MQQPVVRVGEWLVTPSINQISRNGRQLTLEPRLIDLLVFFAQHSGEVLSRDELIDNVWKRSIVTNHVVTQSISELRKSLKDNDEDSPVYIATVPKRGYKLMVPVIWY

[0055] In some embodiments, the TcpP polypeptide of the present invention is fused to the CadC transcription activator DNA-binding domain of E. coli via a linker.

[0056] In some embodiments, the linker consists of the amino acid sequence shown in SEQ ID NO: 46. Sequence ID 46 SEEEGEEIMLSSPPPIPEAVPATDSPSHSLNIQNTATPPEQSPVKSKR

[0057] In some embodiments, the fusion protein of the present invention comprises the amino acid sequence shown in SEQ ID NO: 47. - X 47 This represents N, D, W, Y, T, V, or F. - X 48 This indicates Y or F. - X 49 This represents E, G, V, I, L, S, or K. - X 50 represents Q, V, H, A, T, D, L, or S. Sequence ID 47 MQQPVVRVGEWLVTPSINQISRNGRQLTLEPRLIDLLVFFAQHSGEVLSRDELIDNVWKRSIVTNHVVTQSISELRKSLKDNDEDSPVYIATVPKRGYKLMVPVIWY-SEEEG EEIMLSSPPPIPEAVPATDSPSHSLNIQNTATPPEQSPVKSKR-VVPYLVFSALYVALLPVIWWS-YGQWYQHELAGITHDLRDLARLPGITIQKLSEQKLTFAIDQHQCSV-X 47 -X 48 -X 49 -X 50 -KTLECTKN

[0058] In some embodiments, the fusion protein of the present invention comprises the amino acid sequences shown in SEQ ID NOs. 48-57 disclosed in Table C. [Table 3] JPEG0007854401000004.jpg185159

[0059] The polypeptides disclosed herein, but not limited to those described below, can be prepared by any technique known in the art, such as any chemical, biological, genetic, or enzymatic technique, either alone or in combination. Those skilled in the art can readily prepare the polypeptides described above by standard methods for polypeptide preparation by knowing the amino acid sequence of the desired sequence. For example, this can be synthesized using a commercially available peptide synthesizer (such as one manufactured by Applied Biosystems (Foster City, California)) using a well-known solid-phase method, in accordance with the manufacturer's instructions. Alternatively, the polypeptides and fusion proteins of the present invention can be synthesized by recombinant DNA techniques, as are now well known in the art. For example, these fragments can be obtained as DNA expression products after incorporating a DNA sequence encoding the desired (poly)peptide into an expression vector, introducing such vector into a suitable eukaryotic or prokaryotic host expressing the desired polypeptide, and subsequently isolating it using well-known techniques.

[0060] (Polynucleotides) A further object of the present invention relates to a polynucleotide encoding the bile salt domain of the present invention.

[0061] A further object of the present invention relates to a polynucleotide encoding the TCPP polypeptide of the present invention.

[0062] A further object of the present invention relates to polynucleotides encoding the fusion protein of the present invention.

[0063] A further object of the present invention relates to an expression cassette comprising a polynucleotide operably linked to a regulatory sequence that encodes the fusion protein of the present invention and enables its expression in a prokaryotic host cell.

[0064] Appropriate gene expression regulatory sequences include promoters applicable to the target host organism. Such promoters for diverse prokaryotic hosts are well known to those skilled in the art and are documented in the literature. For example, such promoters can be isolated from naturally occurring genes or can be synthetic or chimeric promoters. Similarly, promoters may already be present in the target genome and can be ligated to polynucleotides by appropriate techniques known in the art, such as homologous recombination.

[0065] In some embodiments, the promoter is selected from the group consisting of a p14 promoter, a p10 promoter, or a p9 promoter, each having the nucleic acid sequences shown in SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60, respectively. Sequence ID 58 > P14 TTGACAATTAATCATCCGGCTCGTATAATGTGTGGA Sequence ID 59 > P10 TTTCAATTTAATCATCCGGCTCGTATAATGTGTGGA Sequence ID 60 > p9 TTGCCTCTTAATCATCGGCTCGTATAATGTGTGGA

[0066] The expression cassette in the present invention is intended for convenient insertion into target polynucleotides, such as vectors or genomic DNA. For this purpose, the expression cassette preferably comprises nucleotide sequences in its 5' and 3' adjacent regions that facilitate removal from or insertion into specific sequence locations, such as restriction enzyme recognition sites or target sequences for homologous recombination catalyzed by, for example, recombinases.

[0067] (Vectors and host cells) A further object of the present invention relates to vectors, particularly plasmids, cosmids, viruses, and bacteriophages, that contain the polynucleotides and expression cassettes of the present invention and are conventionally used in genetic engineering.

[0068] In some embodiments, the vectors of the present invention are suitable for the transformation of prokaryotic host cells. Methods well known to those skilled in the art can be used to construct recombinant vectors. In addition to the polynucleotides or expression cassettes of the present invention, the vectors may include further genes, such as marker genes, that enable the selection of the above-mentioned vectors under appropriate conditions in appropriate host cells. Generally, vectors also include one or more origins of replication. In genetic engineering, for example in prokaryotic host cells, the polynucleotides of the present invention or a portion of these molecules can be introduced into plasmids. Expression vectors are widely described in the literature. Generally, they include not only a selection marker gene and an origin of replication that ensures replication in the selected host, but also a bacterial promoter and often a transcription termination signal. Generally, there is at least one restriction site or polylinker between the promoter and the termination signal, into which an encoding nucleotide sequence can be inserted. It is possible to use promoters that ensure constitutive expression of genes and inducible promoters, enabling intentional control of gene expression. Bacterial promoter sequences having these characteristics are described in detail in the literature. Regulatory sequences for expression in microorganisms (e.g., E. coli) are well described in the literature. Inducible promoters are also possible. This promoter often yields higher protein yields than homeostatic promoters.

[0069] A further object of the present invention relates to a method for producing a prokaryotic host cell capable of expressing the fusion protein of the present invention, the method comprising genetically manipulating the cell with the polynucleotide, expression cassette, or vector of the present invention as described above.

[0070] A further object of the present invention relates to prokaryotic host cells genetically modified with the polynucleotides, expression cassettes, or vectors of the present invention described above, and to cells derived from such transformed cells that contain the polynucleotides, expression cassettes, or vectors of the present invention, and to cells obtained by the method for producing the same described above.

[0071] In some embodiments, the prokaryotic host cell is selected from among Gram-positive bacteria or Gram-negative bacteria.

[0072] In some embodiments, the prokaryotic host cell is selected from among non-pathogenic bacteria. In some embodiments, the prokaryotic host cell is selected from among bacteria originating from a normal internal ecosystem such as a bacterial flora. In some embodiments, the prokaryotic host cell is selected from among non-pathogenic bacteria originating from a normal internal ecosystem of the digestive tract. Non-pathogenic bacteria that are part of the normal flora of the gastrointestinal tract include non-exclusive examples of the genera Bacteroides, Clostridium, Fusobacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, Bifidobacterium, Escherichia, and Lactobacillus.

[0073] In some embodiments, the prokaryotic host cell is selected from among anaerobic bacterial cells (e.g., cells that do not require oxygen for growth). Anaerobic bacterial cells include obligate anaerobic cells such as Bacteroides and Clostridium species. In humans, for example, anaerobic bacterial cells are most commonly found in the digestive tract.

[0074] In some embodiments, the prokaryotic host cell is selected from food-grade bacteria. In some embodiments, the prokaryotic host cell is a probiotic.

[0075] In some embodiments, the prokaryotic host cell is E. coli.

[0076] In some embodiments, prokaryotic host cells are genetically engineered to contain introduced polynucleotides stably integrated into their genome. Transformation of prokaryotic host cells with polynucleotides or vectors in this invention can be carried out by standard methods. For example, gene transfer using calcium chloride can be commonly used in prokaryotic host cells. The prokaryotic host cells are cultured in a nutrient medium that meets the specific requirements of the prokaryotic host cell being used, particularly in terms of pH, temperature, salt concentration, aeration, antibiotics, vitamins, and trace elements.

[0077] In some embodiments, the prokaryotic host cell contains a polynucleotide encoding a TcpH polypeptide having the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the aforementioned polynucleotide is ligated to a promoter p5 having the nucleic acid sequence shown in SEQ ID NO: 61. Sequence ID 61 > P5 TTGACAATTAATCATCCGGCTCGTAATTTATGTGGA

[0078] In some embodiments, the prokaryotic host cell of the present invention comprises at least one further polynucleotide encoding an output molecule whose expression is controlled by the fusion protein of the present invention.

[0079] In particular, the binding of bile salts to the fusion protein induces its oligomerization, thereby oligomerizing the CadC transcription activator DNA-binding domain, which can then activate the expression of at least one further polynucleotide encoding an output molecule under the control of the CadBA promoter.

[0080] Therefore, the prokaryotic host cell of the present invention further comprises a polynucleotide encoding an output molecule ligated to the CadBA promoter. An example nucleic acid of the CadBA promoter is shown by Sequence ID No. 62. Sequence ID 62 >PCadBA ATCCATTGTAAACATTAAATGTTTATCTTTTCATGATATCAACTTGCGATCCTGATGTGTTAATAAAAAACCTCAAGTTCTCACTTACAGAAACTTTTGTGTTATTTCACCTAATCTTTTAGGATTAATCCTTTTTTCGTGAGTAATCTTATCGCCAGTTTGG

[0081] In some embodiments, the output molecule is a polypeptide.

[0082] In some embodiments, the output molecule is a detection protein that can be detected by biological or physical means.

[0083] In some embodiments, the detection protein is a fluorescent protein. The emergence of fluorescent proteins has enabled non-invasive intracellular labeling that is easily detectable by optical means. Green fluorescent protein (GFP) from the jellyfish Aequorea Victoria is currently the most widely used reporter gene in many organisms. Multiple variants with various spectral characteristics have been developed. In some embodiments, prokaryotic host cells contain various combinations of fluorescent proteins that exhibit energy transfer giving fluorescence to be identified. In some embodiments, the detection protein is selected from among luminescent proteins. Certain bacteria (e.g., Vibrio fischeri) have a self-inducible luminescence gene that expresses luciferase, which cleaves luciferin and emits blue light. The bacteria produce a signaling molecule, N-acyl homoseine lactone (AEL), which enters the bacterial cell and induces transcriptional activation of the gene LuxI, which encodes AHL synthetase, and LuxR, which encodes an AHL-dependent transcriptional activator. When the intracellular AHL concentration is sufficiently high, binding to the LuxR activator and transcription of the luminescence gene are triggered.

[0084] Alternatively, the detection protein can be a fusion protein (e.g., green fluorescent protein-Fv) that has detectable properties and is secreted from cells. Therefore, secretion can be induced by a bile salt that binds to the fusion protein of the present invention. In this case, the detection protein is produced in excess, rather than in proportion to the binding of the bile salt.

[0085] In some embodiments, detection can be performed using an RNA aptamer that specifically binds to a fluorescent probe. When the probe binds to the aptamer, its fluorescence increases, enabling the detection of gene expression.

[0086] In some embodiments, the output molecule is a transcription factor that induces the expression of a detectable molecule or a therapeutic molecule. In some embodiments, the output molecule is a repressor factor that suppresses the expression of a detectable molecule or a therapeutic molecule.

[0087] In some embodiments, the output molecule is an endonuclease. In some embodiments, the desired transgene product is an endonuclease that performs site-specific knockdown of gene function, for example, the endonuclease knocks out an allele associated with a genetic disorder. For example, if a dominant allele codes for a defective copy of a gene that is a structural protein and / or functions normally in the wild type, a site-specific endonuclease can target the defective allele and knock it out. In addition to knocking out the defective allele, the site-specific nuclease can also be used to stimulate homologous recombination with donor DNA that codes for a functional copy of the protein encoded by the defective allele. Therefore, for example, a prokaryotic host cell of the present invention can be used both to deliver a site-specific endonuclease that knocks out the defective allele and to deliver a functional copy of the defective allele, resulting in the repair of the defective allele and thereby the production of a functional protein. In some embodiments, the DNA-targeted endonuclease of the present invention is a TALEN. In some embodiments, the DNA-targeted endonuclease of the present invention is a ZFN. In some embodiments, the DNA-targeted endonucleases of the present invention are CRISPR-related endonucleases. In bacteria, the CRISPR / Cas locus encodes an RNA-guided adaptive immune system against mobile genetic elements (viruses, translocation elements, and conjugate plasmids). Three types (I-VI) of CRISPR systems have been identified. A CRISPR cluster contains a spacer, which is a sequence complementary to the preceding mobile element. The CRISPR cluster is transcribed into mature CRISPR (clustered, regularly arranged, short palindromic repeat sequences) RNA (crRNA) and processed. The CRISPR-related endonucleases Cas9 and Cpf1 belong to the type II and type V CRISPR / Cas systems and possess potent endonuclease activity that cleaves target DNA.Cas9 is guided by a mature crRNA containing a specific target sequence of approximately 20 nucleotides (called a spacer), and a transactivatable small RNA (tracrRNA) that acts as a guide for ribonuclease III-assisted processing of the precursor crRNA. The crRNA:tracrRNA double strand guides Cas9 to the target DNA via complementary base pairs between the crRNA spacer and the complementary sequence of the target DNA (called a protospacer). Cas9 recognizes the trinucleotide (NGG) protospacer adjacent motif (PAM) to identify the cleavage site (the third or fourth nucleotide from the PAM). crRNA and tracrRNA can be expressed separately or introduced into artificial fusion small guide RNA (sgRNA) via a synthetic stem-loop to mimic the natural crRNA / tracrRNA double strand. Such sgRNAs, like shRNAs, can be synthesized or transcribed in vitro for direct RNA gene delivery, or expressed from U6 or H1-promoting RNA expression vectors. In some embodiments, the CRISPR-related endonuclease is the Cas9 nuclease. The Cas9 nuclease may have the same nucleotide sequence as the wild-type Streptococcus pyrogenes sequence. In some embodiments, the CRISPR-related endonuclease may be a sequence from another species, e.g., another streptococcal species, e.g., Thermophilus, Pseudomonas aeruginosa, Escherichia coli, or other sequenced bacterial genomes and archaea, or other prokaryotic microorganisms. Alternatively, the Cas9 sequence of wild-type Streptococcus pyrogenes may be modified. The nucleic acid sequence may be codon-optimized, i.e., "humanized," to make it effective for expression in mammalian cells. In some embodiments, the CRISPR-related endonuclease is the Cpf1 nuclease.

[0088] In some embodiments, the output molecule is a therapeutic molecule, particularly a therapeutic polypeptide or therapeutic polynucleotide.

[0089] In the sense of the present invention, therapeutic polypeptides are either naturally occurring proteins such as unmodified growth factors, or designed therapeutic proteins such as single-chain variable fragments of naturally occurring proteins or their variants. Therapeutic polypeptides exert their biological activity through various healing mechanisms. Therapeutic polypeptides are not only growth factors but also other biologically active proteins, such as, but not limited to, protease inhibitors or immunoreceptor antagonists. Therapeutic polypeptides used in this innovation may have the same amino acid sequence and secondary and tertiary structure as naturally occurring ones, or they may be modified or designed to enhance their action. For example, chimeric proteins can be formed by the fusion of various therapeutic polypeptides. Therapeutic polypeptides are also naturally occurring bioactive molecules, such as single-chain variable fragments, recombinant antibodies, peptides that act as antagonists, antibodies (e.g., neutralizing antibodies), nanobodies, or soluble receptors. In some embodiments, the therapeutic polypeptide is a tumor necrosis factor (TNF) or TNF receptor-binding protein, an integrin or integrin receptor-binding protein, or fibroblast growth factor 19 (FGF19). Examples of proteins that bind TNF or TNF receptors include adalimumumab, certolizumab, golimumab, and infliximab, as well as anti-TNF nanobodies.

[0090] In some embodiments, the output molecule is a polynucleotide, particularly a therapeutic molecule. In some embodiments, the output molecule is ribonucleic acid (RNA). In some embodiments, the output molecule is interfering RNA (RNAi).

[0091] Other types of output signals include pigment production via specific operons (such as the violacein operon, or the expression of flavin monooxidase, which converts tryptophan to indigo), or by the expression of enzymes that convert exogenously supplied substrates into chromogenic products, such as beta-galactosidase and its substrate, X-gal.

[0092] Using techniques developed in the field of cellular computation, it is possible to create more complex prokaryotic host cells with higher levels of functionality. In these methods, the cell functions as a biochemical computer, processing inputs such as bile salt binding using internal logic gates to produce outputs. Complex conditioned responses to multiple inputs are manipulated, for example, by executing AND, NOT, OR, XOR, and IMPLIES logic gates in E. coli cells. For example, DNA-binding proteins can be used to execute these gates and modulate the expression of recombinant vectors. Other systems can be used, including but not limited to recombinase-based logic gates, nucleic acid-based logic gates, or protein-based logic gates. For further information on cell computing, all references herein are made to the following: R. Weiss, "Cellular Computation and Communications using Engineered Genetic Regulatory Networks," Ph.D. Thesis, MIT, 2001; MLSimpson et al., "Whole-cell biocomputing," Trends Biotechnol. 19:317-323 (2001); Yaakov Benenson, "Biomolecular computing systems: principles, progress and potential," Nature Reviews Genetics, 13(7):455{468, 2012; Bonnet et al., "Amplifying genetic logic gates," Science, 340(6132):599{603, 2013; Brophy JAN and Voigt CA, "Principles of genetic circuit design," Nature Methods, 11(5):508{520, 2014.

[0093] (Diagnostic method) The prokaryotic host cells of the present invention constitute a whole-cell biosensor ("BactSensor") suitable for the detection and quantification of bile salts, particularly primary bile salts.

[0094] Therefore, a further object of the present invention relates to a method for detecting the presence of bile salts in a sample, the method comprising (i) preparing at least a host cell of the prokaryote of the present invention, (b) contacting the above-mentioned prokaryotic host cell with a sample suspected to contain the above-mentioned bile salts for a time sufficient to allow oligomerization of the binding fusion protein and subsequent expression of the detection protein, and (c) detecting the expression level of the detection protein, the expression level of which correlates with the amount of bile salts present in the sample.

[0095] In some embodiments, the sample is a bodily fluid sample. In some embodiments, the sample is selected from the group consisting of blood samples (including serum or plasma samples), urine samples, cerebrospinal fluid samples, tear samples, saliva samples, and synovial fluid samples.

[0096] The method of the present invention makes it possible to measure the concentration of bile salts dissolved in a sample over a molar range of several orders of magnitude.

[0097] The detection protein is detected for quantification by assaying against bile salts, particularly primary bile salts. Typically, when the detection protein is a fluorescent protein, the fluorescence intensity in each cell can be read by methods known in this technique, such as flow cytometry, laser scanning cytometry, or imaging microscopy. In this way, fluorescence intensity across all desired wavelength ranges can be detected in each individual cell. The amount or concentration of bile salts in the sample can be measured using standard methods. In some embodiments, a calibration curve is constructed by measuring the expression (i.e., its fluorescence) of the detection protein when a sample containing a known concentration of bile salts is combined with cells. The response does not need to be linear as long as a reproducible curve can be constructed. The calibration curve can also be used to correlate the fluorescence intensity of the detection protein measured during the assay with the concentration of bile salts in the sample.

[0098] Those skilled in the art will understand that the methods of the present invention can be used for the detection, identification, and quantification of bile salts in biological and non-biological samples, for example, in the diagnosis of diseases in medicine or veterinary science. These applications can be for commercial purposes (in the sense of routine analysis) or contribute to purely research purposes. Since the methods of the present invention can be used with virtually unlimited modalities, specific detection of thousands of different bile salts becomes possible. Whole-cell sensors can be reused as long as they are not destroyed. In particular, the whole-cell sensors of the present invention are used in in vitro assays as medical diagnostic methods and disease management.

[0099] In particular, the whole-cell sensor of the present invention is especially suitable for diagnosing liver dysfunction in subjects.

[0100] Accordingly, a further object of the present invention relates to a method for determining whether a subject has or is at risk of having hepatic impairment, the method comprising (i) preparing at least a host cell of the prokaryote of the present invention, (b) contacting the above-mentioned prokaryotic host cell with a sample obtained from the subject for a time sufficient to allow oligomerization of the binding fusion protein and subsequent expression of the detection protein, and (c) detecting the expression level of the detection protein, the expression level of which correlates with the amount of bile salts present in the sample, the amount of which the bile salts indicates whether the subject has or is at risk of having hepatic impairment.

[0101] Numerous acute or chronic pathological conditions can lead to liver dysfunction. These include, but are not limited to, liver abscess, primary or metastatic liver cancer, cirrhosis, e.g., cirrhosis caused by alcohol consumption or primary biliary cirrhosis, amoebic liver abscess, autoimmune hepatitis, biliary atresia, disseminated coccidioidomycosis, portal hypertension, liver infections (e.g., hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, or hepatitis E virus), hemochromatosis, hepatocellular carcinoma, suppurative liver abscess, Reye's syndrome, sclerosing cholangitis, Wilson's disease, drug-induced hepatotoxicity, or fulminant hepatic failure or acute hepatic failure. In some embodiments, the liver disease is non-alcoholic fatty liver disease. In some embodiments, the liver disease is drug-induced liver disease. In some embodiments, the liver disease is alcoholic liver disease.

[0102] In some embodiments, liver dysfunction may result from viral infection. The liver is involved in infections by liver-targeting viruses that replicate in and primarily target the liver, for example. These include hepatitis A virus, hepatitis B virus, hepatitis C virus, and hepatitis E virus. In all of these infections, hepatitis and liver dysfunction result from immune responses and repair mechanisms (e.g., fibrosis) against the virus within the liver. Furthermore, the liver may be affected as part of a systemic host infection by viruses that primarily target other tissues, particularly the upper respiratory tract. Examples of viruses include herpesviruses (Epstein-Barr virus, cytomegalovirus [CMV], and herpes simplex virus), parvoviruses, adenoviruses, and severe acute respiratory syndrome (SARS)-associated coronaviruses (e.g., SARS-CoV-2).

[0103] In some embodiments, the diagnostic methods described herein are applied to subjects exhibiting symptoms of hepatic dysfunction who have not undergone routine screening to rule out all possible causes of hepatic dysfunction. The methods described herein may be part of a routine set of tests performed on subjects exhibiting symptoms of hepatic dysfunction such as jaundice, abdominal pain and bloating, swelling of the legs and ankles, itchy skin, dark urine, pale stools, bloody stools, black stools, chronic fatigue, nausea or vomiting, loss of appetite, easy bruising, etc. The methods of the present invention may be performed in addition to other diagnostic tools including ultrasound evaluation (e.g., elastography), biopsy, and / or quantification of at least one further biomarker such as levels of AST, ALT, ALP, TTT, ZTT, total bilirubin, total protein, albumin, lactate dehydrogenase, and cholinesterase in blood.

[0104] In some embodiments, the subjects are those who have undergone liver transplantation. Therefore, the present invention is particularly suitable for determining whether a liver transplant subject has graft rejection or is at risk of having graft rejection.

[0105] In some embodiments, the methods of the present invention are particularly suited to determining whether a subject suffering from liver disease will respond to treatment. The methods are therefore particularly suited to distinguishing responders from non-responders. In the context of this disclosure, responders refer to subjects capable of responding, i.e., subjects in remission, and more specifically, subjects not suffering from liver dysfunction. Non-responders include subjects whose disease does not show reduction or improvement after treatment (e.g., liver dysfunction remains stable or decreases). In the present invention, treatment is based on any method or agent that may be appropriate for treating liver dysfunction. Some liver problems can typically be treated by lifestyle changes, such as discontinuing alcohol use or weight loss, as part of a medical program that includes careful monitoring of liver function. Each liver disease may have its own specific treatment regimen. For example, hepatitis A requires supportive nursing care to maintain hydration while the body's immune system fights and clears the infection. Patients with gallstones may require surgery to remove the gallbladder. Other diseases may require long-term medical care to manage and minimize the consequences of those diseases. In patients with cirrhosis and end-stage liver disease, medication may be necessary to regulate the amount of protein absorbed from the diet. Other examples include surgery required to treat portal hypertension.

[0106] The method of the present invention is particularly suitable for monitoring the effectiveness of treatment. Typically, a decrease in binding capacity (e.g., between measurements taken at different time intervals) indicates that the subject is not responding to the treatment. Conversely, an increase in binding capacity (e.g., between measurements taken at different time intervals) indicates that the subject is responding to the treatment.

[0107] Furthermore, the method of the present invention is particularly suitable for evaluating the effects of a drug under development by causing liver injury during preclinical or clinical research.

[0108] (Biosensor) Furthermore, the whole-cell sensor of the present invention can be converted into a biosensor device that can be formed using the whole-cell sensor of the present invention for deployment in a microenvironment or microfluidic device, or in a group of such devices in a multi-chip module or distributed wireless network. The biosensor device can respond to one or more specific chemical and / or physical inputs (e.g., heat or electric current), produce an output in the form of a detection protein, and communicate with a physical transducer by means of calorimetry, electrochemistry, or preferably fluorescence bioluminescence. The biosensor can therefore include a detection component containing whole-cell sensors, and a transducer component for converting the physical or chemical changes produced by the detection component into electrical signals. In a mechanism for biomarker detection, there are five types of transducers that can be used in the biosensor of the present invention: optical (colorimetric, fluorescence, emission, and interference) transducers, mass-based (piezoelectric, and elastic wave) transducers, magnetic field-based transducers, electrochemical (current measurement, potentiometric, and conductivity measurement) transducers, and calorimetry transducers. In some embodiments, the device may include further measuring devices for measuring parameters of other purposes. Typically, the system may include further devices suitable for measuring physiological phenotypes. Physiological phenotypes may include physiological parameters such as body temperature, pulse rate, blood pressure, respiratory rate, and hydration status. In some embodiments, the system may include an input / output module, an analysis module, and a report generation module. The input / output module is configured to receive bile salt amounts, optionally combined with further parameters, via an optionally accompanying communication device. The analysis module is configured to analyze various parameters, including bile salt amounts. The report generation module is configured to generate a profile of the subject in the analysis of various parameters.In some embodiments, the system includes a sharing module that shares one or more pre-formatted messages with one or more stakeholders based on a comparison of the generated subject profiles. In some embodiments, the system includes providing recommendations to a subject to alert them about the risk of having liver dysfunction. In some embodiments, the system includes enabling the transmission of another message to an external worker (e.g., a physician) to alert them that the subject has or is at risk of having liver dysfunction. Thus, in some embodiments, the device includes a communication device. Examples of communication devices include, but are not limited to, mobile phones, tablets, and desktop computers. Various media, including the Internet, intranets, Bluetooth®, and Wi-Fi, can be used for connection. In some embodiments, the communication device is connected to a server. Measurement values ​​of one or more parameters measured by the measuring device can actually be transmitted wirelessly to a handheld device including a microprocessor. The handheld device can be a smartphone, tablet device, mobile phone, mobile internet device, netbook, notebook, personal digital assistant, internet phone, hologram device, hologram phone, cable internet device, satellite internet device, internet television, DSL internet device, and remote control.

[0109] (kit) A further object of the present invention relates to a kit for carrying out the methods disclosed herein. The kit comprises one or more whole-cell sensors as described above, and means for measuring the expression level of the detection protein. Reagents for specific types of assays can also be provided within the kit of the present invention. In some embodiments, the kit includes devices such as biosensors as described above. Furthermore, the kit may include various diluents and buffers, labeled conjugates or other agents for detecting specific immune complexes. Other components in the kit can be readily determined by those skilled in the art.

[0110] (Treatment method) Furthermore, prokaryotic host cells are particularly suitable for therapeutic purposes. In particular, the manipulated prokaryotic host cells in this invention are suitable for functioning in the intestines and can be specifically activated upon reaching the intestinal microenvironment where bile salts are present. The aforementioned prokaryotic host cells are particularly suitable for expressing therapeutic molecules (polynucleotides or polypeptides) in the intestines. The expression of output therapeutic molecules can be induced in the presence of bile salts, especially primary bile salts.

[0111] Therefore, a further object of the present invention relates to a method of treatment in a subject requiring treatment, the method comprising administering a therapeutically effective amount of a prokaryotic host cell of the present invention containing a polynucleotide encoding a therapeutic molecule to the subject.

[0112] Examples of diseases that can be treated by the methods of the present invention include, but are not limited to, obesity, inflammatory bowel disease, colorectal cancer, liver disease, and hepatobiliary tract disease. In some embodiments, this disease or disorder is peptic ulcer disease, cirrhosis, inflammatory bowel disease, infection, cancer, vascular disorders, adverse drug reactions, or blood coagulation disorders. In some embodiments, the subject has or is at risk of having inflammatory bowel disease. Inflammatory bowel disease (IBD) refers to a group of inflammatory conditions of the small and colon. In some embodiments, IBD is Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, vacuum colitis, Behçet's disease, or atypical colitis.

[0113] In some embodiments, prokaryotic host cells are administered into the intestines.

[0114] In some embodiments, the prokaryotic host cells of the present invention are encapsulated for protection against the stomach. Therefore, in some embodiments, the prokaryotic host cells of the present invention are formulated in an encapsulated form in the composition to significantly improve their survival time. In such cases, the presence of the capsule can particularly delay or inhibit the degradation of the prokaryotic host cells in the gastrointestinal tract. It can be understood that the compositions of this embodiment can be encapsulated in enteric-coated sustained-release capsules or tablets. The enteric coating allows the capsule / tablet to remain unchanged (i.e., undissolved) as it passes through the gastrointestinal tract until it reaches the intestinal tract. Methods for encapsulating viable bacterial cells are well known in the art (see, for example, U.S. Patent No. 6,723,358 of General Mills Inc.). For example, encapsulation can preferably be carried out with an enteric coating, which is a methacrylate-alkyl acrylate copolymer such as Eudragit® polymer. Poly(meth)acrylates have been found to be particularly suitable as coating materials.

[0115] In some embodiments, the host cells of the prokaryotes of the present invention are administered to a subject in the form of a food composition. In some embodiments, the food composition is selected from complete food compositions, food supplements, and nutraceutical compositions, etc. The compositions of the present invention can be used as food components and / or feed components. Food components can be in the form of a solution or a solid, depending on the mode of use and / or application and / or administration. Food compositions and nutritional supplement compositions are, for example, fermented dairy products or dairy-based products that are administered or ingested orally, preferably once or more per day. Fermented dairy products can be produced by directly using the bacteria of the present invention in the manufacturing process, for example, by adding them to food components using methods known to the present invention. In such methods, the strains of the present invention may be used in addition to and / or replace one or more or some of the commonly used microorganisms. Fermented dairy products include milk-based products, such as (but not limited to) desserts, yogurt, yogurt drinks, quark, kefir, fermented milk-based beverages, buttermilk, cheese, dressings, low-fat spreads, fresh cheese, soy-based beverages, and ice cream. Alternatively, food compositions and / or food supplement compositions may be non-dairy products or non-fermented dairy products (e.g., non-fermented milk or bacterial strains or cell-free culture media in other food media). Non-fermented dairy products may include ice cream, nutritional supplement bars, and dressings. Non-dairy products may also include powdered beverages and nutritional supplement bars.

[0116] In some embodiments, the food composition containing the prokaryotic host cells of the present invention contains at least one prebiotic, i.e., a food substance used to promote the proliferation of the prokaryotic host cells of the present invention in the intestinal tract. The prebiotic can be selected from the group consisting of oligosaccharides and optionally contains fructose, galactose, mannose, soy, and / or inulin, and / or dietary fiber.

[0117] In the context of this invention, the amount of prokaryotic host cells administered to a subject may depend on the subject's general health status, age, sex, weight, and other individual characteristics. Those skilled in the art can determine an appropriate dosage based on these and other factors. For example, a sufficient amount of prokaryotic host cells capable of producing colonies may be enough to produce a beneficial effect on the subject. When prokaryotic host cells are administered in the form of a food product, typically, 10 per gram of dry weight of the food composition is used. 3 ~10 12 The CFU may include a prokaryotic host cell according to the present invention.

[0118] (Screening method) Furthermore, the prokaryotic host cells of the present invention are particularly suitable for screening purposes. In particular, the prokaryotic host cells of the present invention are particularly suitable for screening drugs that are suitable for inhibiting pathogen signaling pathways, for example. In this case, the system is a rewired version of the Vibrio cholera pathogenicity activation pathway. When constructed in non-pathogenic prokaryotic host cells such as E. coli and easily monitored by detecting an output molecule capable of detecting activation, the system can function as a high-throughput screening platform for compound libraries to identify novel inhibitors (or activators) of V. cholera pathogenicity that could be used as therapeutic agents.

[0119] Therefore, a further object of the present invention relates to a method for screening multiple test substances, the method comprising (i) contacting a population of host cells of the prokaryotic organism of the present invention with the above-mentioned test substances in the presence of a predetermined amount of bile salt, and (ii) selecting test substances that can modulate the expression of output molecules.

[0120] The test substance of the present invention can be selected from a library of substances synthesized to date, a library of substances whose structures have been determined in a database, or a library of substances synthesized de novo. The test substance can be selected from the group consisting of (a) proteins or peptides, (b) nucleic acids, and (c) organic substances or chemical substances.

[0121] In some embodiments, the method includes the steps of comparing the expression level of an output molecule (e.g., a detection protein) with the expression level measured in the absence of the test substance, and making a positive selection of a test substance that results in a decrease or increase in the expression level of the output molecule.

[0122] The present invention will be further described with reference to the following drawings and embodiments. However, these embodiments and drawings should not be construed in any way as limiting the scope of the present invention. [Examples]

[0123] (Example 1: TCPP sensor) The liver is a vital organ that coordinates metabolic, detoxification, and immunological processes. Liver diseases, including hepatitis, cirrhosis, fatty liver disease, and cancer, are major public health issues requiring large-scale screening methods for prevention, diagnosis, and therapeutic monitoring. Liver function is typically monitored by quantifying serum enzyme activity and bilirubin, but these markers are not entirely specific and are only detectable when the impairment has already progressed. Liver function is also typically monitored by quantifying multiple enzyme activities together, as these are not specific to each other. Serum and urinary bile salts are another biomarker for the early diagnosis of liver dysfunction, but current detection methods are impractical and their measurement is difficult.

[0124] In this specification, we designed a bacterial biosensor based on non-pathogenic E. coli to detect the concentration of pathological bile salts in clinical samples. The TcpP bile salt sensing domain of Vibrio cholera, a bacterial component that controls the activation of the pathogenic operon when pathogens enter the intestine, was used for a different purpose. A synthetic bile salt receptor was designed using TcpP as a sensing domain conjugated to the CadC system of E. coli, which activates transcription upon dimerization (Figure 1). The system's performance was assayed against various selected promoters (Figures 2A and 2B), demonstrating that a fine-tunable response can be achieved by altering the expression level of the bile salt receptor. The system was also tested with various bile salts (Figure 3). The response of the bactosensor to a group of 12 different bile salts, including both primary and secondary types, was measured. Interestingly, while the sensing module was not specific to a single bile salt species, the CadC-TcpP system was highly specific to conjugated primary bile salts and did not respond to secondary bile salts.

[0125] The aim was to identify key residues that determine the sensitivity of the TcpP sensing module, targeting them to improve the sensitivity and LOD of the synthetic receptor. Therefore, comprehensive mutagenesis, combined with functional screening and next-generation sequencing (NGS), supports the identification of functional mutants along with sequence determinants within localized structural motifs. The transition from intramolecular disulfide bonds to intermolecular disulfide bonds between TcpP monomers is a major determinant of the TcpP response to bile salts, mediated by two cysteine ​​residues, Cys207 and Cys218. Multiple sequence alignments of various TcpP bacterial homologs (data not shown) revealed significant conservation of the amino acid flanked by these two cysteines (data not shown). Secondary structure prediction and 3D prediction of abu initio using the Rosetta modeling suite (data not shown) suggested that each cysteine ​​is located on a rigid β-sheet separated by a flexible loop region between Asn211 and Gln214. The nature of this loop, which forms a turn, allows the two β-sheets and cysteine ​​to come into close proximity, potentially forming an intramolecular disulfide bond. We hypothesized that the flexibility of the turn region between Cys207 and Cys218 is a key parameter controlling the transition rate between the two states, and that altering its amino acid composition can change the system's sensitivity to bile salts.

[0126] Therefore, a comprehensive mutation library targeting NYEK residues within turns (NNK x 4, logical library complexity ≈ 1.05 x 10) is needed. 6 A mutant was constructed and cloned into a plasmid that constitutively expresses CadC-TcpP and generates GFP in response to bile salts (data not shown). The resulting library was induced with TCA, and GFP-positive mutants were isolated by fluorescence-activated cell sorting (FACS). Three rounds of enrichment were performed (200 μM TCA as ligand in the first and second rounds of selection, and 20 μM in the third round), and the increase in fractions of cell populations in response to various ligand concentrations (20-80 μM) was observed (data not shown). Single mutants were isolated, cultured, and sequenced to test their response to TCA (data not shown). It was found that comprehensive mutagenesis of residues from Asn210 to Gln213 could alter the detection limit, sensitivity, and activation ratio of this biosensor. The 3.3-fold difference in LOD between SV_3-3-18 and SV_3-3-22 (EC50 of 28.3–92.5 μM) demonstrated that mutations in the loop region of the TcpP sensing module can lead to a wide range of sensitivity designs. Further kinetic analysis revealed that this variation in the loop region alters the reaction rate with the bile salt synthesis receptor and the system's interaction. The mutant SV_3-3-18 exhibits a 13-fold increase in ligand affinity and responds more quickly at low ligand concentrations compared to wild-type TcpP (data not shown).

[0127] To better understand the sequence features that influence the TcpP response to bile salts, the entire enriched mutant pool was sequenced by next-generation sequencing (NGS). Surprisingly, the sequence features of the functional mutants differed from those expected from the natural TcpP homolog (data not shown). First, in contrast to the wild-type TcpP homolog, we observed a high degree of deletion of long-chain polar amino acids (Asn and Gln) along with long-chain loaded amino acids (Asp and Glu) at position 211. Furthermore, the functional mutants appeared to be deleting lysine at position 211 (which is commonly found at this position in other TcpP homolog proteins). Next, amino acids with large aromatic side chains, such as Phe and Tyr, and amino acids with hydrophobic side chains, such as Leu, were highly conserved in the selected functional mutants, strongly indicating the crucial role of the hydrophobic residue at position 211 in the C-terminal loop region in the function of V. cholerae TcpP. The best-engineered mutant was SV_3-3-18.

[0128] By inductively evolving multiple rounds of the TCPP sensor, we obtained a group of mutants with lower detection limits and higher sensitivity (Table 1 and Figure 4). Finally, we demonstrate that this Bact sensor can detect pathological bile salt concentrations in samples from patients with hepatic impairment (Figures 5A and 5B). Table 1 lists various mutants and their characteristics. [Table 4]

[0129] This research paves the way for a highly sensitive, scalable, and inexpensive screening platform for liver dysfunction that can be deployed in point-of-care or home settings, enabling large-scale monitoring of liver-related diseases. Furthermore, the study demonstrates how synthetic biology can help address global healthcare challenges by providing tools to elucidate and target fundamental cellular mechanisms, in this case, pathogen signaling.

[0130] (Example 2: Colorimetric analysis assay) Colorimetric assays provide a simple and intuitive method for easily and directly evaluating test results by the naked eye. Furthermore, colorimetric assays support the easy development of quantitative assays using smartphone-based platforms for point-of-care (POC) or home diagnostics. Colorimetric analysis outputs were obtained using the SV_3-3-18 mutant (referred to as SV_3-3-18-LacZ) conjugated with the reporter β-galactosidase LacZ and its substrate, chlorophenol red-β-D-galactopyranoside (CPRG) (data not shown). Similar to biosensors with GFP output, the bile salt specificity profile of the SV_3-3-18-LacZ system shifted slightly from TCA to GCDCA (Figure 6). Therefore, the LOD and signal output thresholds of SV_3-3-18-LacZ corresponding to increasing concentrations of GCDCA were evaluated. The effects of various cell densities and incubation times were also investigated (data not shown). Increasing cell density or incubation time both improved the dynamic range and operating range of SV_3-3-18-LacZ, but also increased background signal. After optimization, SV_3-3-18-LacZ showed a linear response to GCDCA within 1 hour at concentrations of 0–40 μM (data not shown). Furthermore, by adjusting cell density and incubation time, its threshold activation level can be matched to various clinical levels associated with specific liver-related conditions.

[0131] (Example 3: Detection of elevated bile salt levels in serum from patients who underwent liver transplantation via a bactosensor) The sensor was tested in samples from patients who underwent liver transplantation. The main complications after liver transplantation are bile duct stenosis and acute cellular rejection.

[0132] The Bactosensor was tested in 21 serum clinical samples from liver transplant patients (data not shown). Most of the patients underwent liver transplants in the past two years, and they were followed up at the Montpellier Hospital after their transplants. These patients underwent liver transplants due to end-stage liver disease resulting from alcohol-related liver disease or non-alcoholic fatty liver disease, chronic cholangitis, or liver cancer. Thorough liver examinations were performed, and serum bile salts were measured using enzyme assays (data not shown). Patients with a high probability of acute cellular rejection (ACR) after liver transplantation (serum bile acid >37 μM) were found to have significant and visible colorimetric analytic signal changes in the Bactosensor assay (data not shown). Three patients showing elevated serum bile salt concentrations were noteworthy. Two of these patients showed abnormal liver enzyme levels (ASAT, ALAT, GGT, Pal, and bilirubin). The bile salt bactosensor produced the strongest colorimetric changes readily detectable with the naked eye in these patients (Figure 7). These results indicate that the bactosensor can provide a simple, reliable, and cost-effective method for monitoring the condition of patients after liver transplantation.

[0133] (References) In this application, the current state of the art to which the present invention belongs is described by various references. The disclosures of these references are incorporated herein by reference.< / ygqwyqhelagithdlrdlarlpgitiqklseqkltfaidqhqcsvnyeqktlectkn>

Claims

1. A bile salt sensing domain comprising the amino acid sequences shown in SEQ ID NOs: 25-33.

2. A TcpP polypeptide comprising the amino acid sequences shown in SEQ ID NOs: 36 to 44.

3. A fusion protein obtained by fusing a TcpP polypeptide with a heterologous polypeptide, wherein the TcpP polypeptide contains the amino acid sequences shown in SEQ ID NOs: 36 to 44.

4. The fusion protein according to claim 3, wherein the heterologous polypeptide is a DNA-binding domain.

5. The fusion protein according to claim 3, wherein the heterologous polypeptide is the CadC transcription activator DNA-binding domain of E. coli.

6. The fusion protein according to claim 3, wherein the TcpP polypeptide is fused with the heterologous polypeptide either directly or via a linker.

7. The fusion protein according to claim 6, wherein the linker consists of the amino acid sequence shown in Sequence ID No.

46.

8. The fusion protein according to claim 6, comprising the amino acid sequences shown in SEQ ID NOs: 48-57.

9. A polynucleotide encoding a bile salt sensing domain according to claim 1, and / or a TcpP polypeptide according to claim 2, and / or a polynucleotide encoding a fusion protein according to claim 3.

10. An expression cassette comprising a polynucleotide encoding the fusion protein described in claim 3 and operably linked to a regulatory sequence that enables expression in a prokaryotic host cell.

11. The expression cassette according to claim 10, comprising a promoter selected from the group consisting of a p14 promoter, a p10 promoter, or a p9 promoter having the nucleic acid sequences shown in SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO:

60.

12. A prokaryotic host cell genetically engineered with the polynucleotide described in claim 9 or the expression cassette described in claim 10.

13. A host cell for a prokaryote according to claim 12, selected from bacteria belonging to the genera Bacteroides, Clostridium, Fusobacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, Bifidobacterium, Escherichia, and Lactobacillus.

14. The host cell of the prokaryote according to claim 12, which is E. coli.

15. A prokaryotic host cell according to claim 12, comprising a polynucleotide encoding a TcpH polypeptide having the amino acid sequence shown in SEQ ID NO: 2, wherein the polynucleotide is optionally ligated to a promoter p5 having the nucleic acid sequence shown in SEQ ID NO:

61.

16. A prokaryotic host cell according to claim 12, comprising at least one further polynucleotide whose expression encodes an output molecule under the control of the fusion protein according to claim 3.

17. The prokaryotic host cell according to claim 12, further comprising a polynucleotide encoding an output molecule ligated to the CadBA promoter of Sequence ID No.

62.

18. The host cell of a prokaryotic organism according to claim 16 or 17, wherein the output molecule is a detection protein such as a fluorescent protein.

19. The prokaryotic host cell according to claim 16 or 17, wherein the output molecule is a therapeutic polypeptide.

20. A method for detecting the presence of bile salts in a sample, comprising: (i) preparing a prokaryotic host cell according to at least one claim 18; (b) contacting the prokaryotic host cell with the sample suspected to contain the bile salts for a time sufficient to allow oligomerization of the binding fusion protein and subsequent expression of the detected protein; and (c) detecting the expression level of the detected protein, wherein the expression level correlates with the amount of bile salts present in the sample.

21. A prokaryotic host cell according to claim 18, used to determine whether a subject has liver dysfunction or is at risk of developing liver dysfunction.

22. A prokaryotic host cell according to claim 19, used for the treatment of a subject requiring treatment.

23. A prokaryotic host cell according to claim 22 for treating obesity, inflammatory bowel disease, colorectal cancer, liver disease, and hepatobiliary disease.

24. A method for screening multiple test substances, comprising: (i) contacting a population of prokaryotic host cells according to claim 18 with the multiple test substances in the presence of a predetermined amount of bile salt; and (ii) selecting the test substances that can modulate the expression of the output molecule.

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