Bacterial composition with modified enzymes and methods of using the same

Abstract

The invention relates to genetically modified Bacteroides thetaiotaomicron expressing one or more heterologous genes encoding at least one bile salt metabolism enzyme, and cells and methods for producing modified enzymes in cells to increase bile acid deconjugation.

Description

[0001] TITLE OF THE INVENTION

[0002] Bacterial Composition with Modified Enzymes and Methods of Using The Same

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Application No. 63 / 700,715, filed September 29, 2024, which is hereby incorporated by reference herein in its entirety.

[0005] REFERENCE TO A “SEQUENCE LISTING” SUBMITTED AS AN XML FILE The present application hereby incorporates by reference the entire contents of the sequence listing xml document named “206653-0001-

[0006] OOWO Sequence Listing.xml”. The xml file containing the Sequence Listing of the present application was created on September 29, 2025 and is 92,881 bytes in size.

[0007] BACKGROUND OF THE INVENTION

[0008] The gastrointestinal tract hosts a large and complex community of microorganisms, collectively known as the gut microbiota. This community consists of around 1014microorganisms and is dominated by the presence of about more than 1000 different bacterial species. The gut microbiota lives in a close relationship with the host and is crucial for many physiological processes. Several mechanisms have been proposed to mediate the effects of these microorganisms on metabolic health. One important function carried out by the human gut microbiota is the deconjugation of primary bile acids (BAs) by Bile salt hydrolase (BSH) enzymes and their impact on host health. The presence of active BSHs has long been recognized as a selection criterion for defining potential probiotics. BAs are amphipathic molecules that are initially produced by the host and subsequently transformed by the gut microbiota. Host primary BAs (CA, cholic acid; CDCA, chenodeoxycholic acid) are synthesized from cholesterol and conjugated with glycine or taurine in the liver (GCA and GCDCA, glycocholic acid and glycochenodeoxycholic acid; TCA and TCDCA, taurocholic acid and taurochenodeoxycholic acid). Both primary and secondary BAs serve as signaling molecules that influence several processes, including lipid, glucose, and energy metabolism as well as inflammation.

[0009] BSH enzymes are widely distributed in human gastrointestinal microbial communities and are believed to play key roles in both microbial and host physiology. As such, deconjugation of BAs by BSH enzymes comprises an imperative gateway reaction in the metabolism of BAs. BSH is produced by the intestinal microbiota that catalyzes the hydrolysis of amide bonds in conjugated BAs, resulting in the release of free amino acids.

[0010] BSH enzymes play important roles in a wide range of host metabolic processes, including the regulation of cholesterol metabolism, energy, and inflammation homeostasis. Functional analysis of the gut microbiota revealed a high number of these enzymes in this ecological niche. The modulation of such activity has been shown to exhibit widespread effects on the host and resident microbiota.

[0011] Although primary and secondary BAs were initially only recognized for their role in lipid digestion, subsequent research revealed that these cholesterol derivatives have a much more complex role in human physiology. Beyond their digestive functions, BAs circulate throughout the body, acting as signaling molecules that influence systemic metabolism. This signaling occurs through the activation of specific receptors, such as the famesoid X receptor (FXR) and the G protein-coupled bile acid receptor 1 (GPBAR1, also known as Takeda G protein-coupled receptor 5 [TGR5]). Additionally, BAs exhibit selective antimicrobial activity, which shapes the gut microbiota. The intricate interactions between BAs, gut microbes, and host BA receptors significantly impact host pathophysiology, highlighting the multifaceted roles of BAs in maintaining health and contributing to disease processes.

[0012] Metabolic disorders occur when the breakdown of food to its components becomes disrupted. Disorders in metabolism can be inherited, in which case they are known as inborn errors of metabolism, or they may be acquired during the lifetime. Metabolic disorders can be inherent to severe diseases or conditions, including respiratory or liver failure, chronic obstructive pulmonary disease, and cancer. Occasionally highly complex pathways mediate metabolic disorders. At other times, one base-pair of the DNA may be solely responsible. These discoveries have led scientists to develop extraordinary treatments for affected individuals, and the pace of discovery continues to accelerate. The symptoms of metabolic disorders vary among individuals and by the type of the disorder. Some metabolic disorders result in mild symptoms that can be managed with treatment and lifestyle changes, whereas others can cause severe and life-threatening symptoms, such as heart issues, breathing problems, and organ failure. Some inherited metabolic disorders can require long-term nutritional supplementation and treatment, however, metabolic disorders that arise as a result of another disease or disorder frequently resolve once the underlying condition is treated. One of the prominent examples is Metabolic syndrome - a highly prevalent disease cluster worldwide. It requires polypharmacological treatment of conditions including type II diabetes, hypertension, and dyslipidemia, as well as the associated comorbidities. The complex treatment regimens with various drugs lead to drug-drug interactions and inadequate patient adherence, resulting in poor management of the disease.

[0013] Another example is the complex relationship between BAs and the microbiome in the development of metabolic dysfunction-associated fatty liver disease (MAFLD). MAFLD / MASLD is characterized by excess fat accumulation or steatosis in the liver and is primarily associated with metabolic conditions such as obesity, type 2 diabetes, and insulin resistance. If untreated, MAFLD / MASLD can progress from steatosis to metabolic dysfunction-associated steatohepatitis (MASH) potentially leading to liver fibrosis, cirrhosis, and even liver failure or cancer. In this respect, a secondary bile acid 3-succinylated cholic acid (c-sucCA) is biosynthesized by bile acid acyl synthetase for succinyl (BAS-suc), an enzyme annotated as (3-lactamase in Bacteroides uniformis. C-sucCA was found to alleviate metabolic dysfunction associated steatohepatitis (MASH) progression by enriching the abundance of the beneficial gut bacterium Akkermcmsia muciniphila.

[0014] As the unique enzymes involved in the crucial deconjugation reaction, BSHs, BAS-suc and others, may serve as a promising strategy to control numerous diseases ranging from metabolic disorders to inflammatory and infectious diseases. Studies highlighting the molecular aspects of BSH enzymes including protein structure and genetic regulation would certainly be of great relevance to fully address and evaluate the implications of BSHs as a clinical tool. Therefore, identifying safe, efficient and holistic solutions and treatment options for the disease conditions associated with impaired metabolism remains a long unmet need.

[0015] Thus, there is a need in the art for more effective therapeutic strategies for enhancing enzymatic activity, lowering enzymatic activity, enhancing substrate specificity and / or enhancing stability, bile acid deconjugation and treating conditions associated with impaired metabolism. This invention satisfies this unmet need.

[0016] SUMMARY OF THE INVENTION

[0017] In some embodiments, the invention provides a modified bile salt hydrolase (BSH) having at least 80% homology to an amino acid sequence set forth as SEQ ID NO: 34, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33 and comprising one or more substitutions relative to SEQ ID NO: 34, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33.

[0018] In some embodiments, the modified BSH comprises at least 90% homology to an amino acid sequence set forth as SEQ ID NO: 34, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33 and comprising one or more substitutions relative to SEQ ID NO: 34, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33.

[0019] In some embodiments, the modified BSH comprises an amino acid sequence set forth as SEQ ID NO: 7, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 4, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0020] In some embodiments, the modified BSH comprises at least one amino acid substitution at position L67, K195, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 31 or 33.

[0021] In some embodiments, the modified BSH comprises a combination of at least two amino acid substitutions at any of the positions L67, K195, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 31 or 33.

[0022] In some embodiments, the modified BSH comprises a combination of multiple amino acid substitutions at any of the positions L67, K195, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 31 or 33. In some embodiments, the modified BSH comprises at least one amino acid substitution at position Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 32.

[0023] In some embodiments, the modified BSH comprises at least one amino acid substitution at position Y34, H47, 157, L127, KI 89, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34.

[0024] In some embodiments, the modified BSH comprises at least two amino acid substitutions at any of the positions Y34, H47, 157, L127, K189, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34.

[0025] In some embodiments, the modified BSH comprises a combination of multiple amino acid substitutions at any of the positions Y34, H47, 157, L127, K189, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34.

[0026] In some embodiments, the modified BSH is expressed in a modified bacterial cell.

[0027] In some embodiments, the modified bacterial cell is Bacteroides thetaiotaomicron .

[0028] In some embodiments, the invention provides an isolated oligonucleotide having at least 80% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 41 to 44.

[0029] In some embodiments, the isolated oligonucleotide comprises at least 90% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 41 to 44.

[0030] In some embodiments, the isolated oligonucleotide comprises a polynucleotide sequence set forth as SEQ ID NO: 16 to 30 or SEQ ID NO: 51 to 55.

[0031] In some embodiments, the isolated oligonucleotide is operably linked to at least one promotor.

[0032] In some embodiments, the invention provides a composition comprises an isolated oligonucleotide, an expression vector, a modified BSH, or any combination thereof, and at least one carrier.

[0033] In some embodiments, the composition further comprises a bile acid acyl synthetase for succinyl (BAS-suc) comprising an amino acid sequence set forth as SEQ ID NO: 35, SEQ ID NO: 50, or a homologue thereof. In some embodiments, the composition is for use as a medicament.

[0034] In some embodiments, the composition is a nutraceutical, supplement, medical food, and / or GRAS designated products.

[0035] In some embodiments, the composition is for use in the treatment of a disease or condition associated with impaired metabolism.

[0036] In some embodiments, the invention provides a method of treating a disease or condition associated with impaired metabolism, comprising administering to the subject in need an effective amount of the composition.

[0037] In some embodiments, the invention provides a method of treating a condition associated with impaired metabolism and / or lipid metabolism, comprising administering to a subject in need of such treatment an effective amount of the composition.

[0038] In some embodiments, the invention provides a method of alleviating at least one symptom of a disease or condition associated with impaired metabolism comprising administering to a subject afflicted with said disease or condition an effective amount of the composition comprising the modified BSH.

[0039] In some embodiments, the disease or condition is associated with impaired lipid metabolism.

[0040] In some embodiments, the invention provides a method of preventing and / or delaying an onset and / or or preventing and / or slowing progression of a disease or condition associated with impaired metabolism comprising administering to a subject an amount of the composition effective to prevent or delay progression of a disease or condition associated with impaired metabolism.

[0041] In some embodiments, the disease or condition is associated with impaired lipid metabolism.

[0042] In some embodiments, the condition associated with impaired metabolism is selected from the group consisting of a metabolic disorder, metabolic syndrome, cardiovascular disease, cholestatic disease, hyperlipidemia, dyslipidemia, insulin resistance, type II diabetes, impaired glucose tolerance, obesity, liver disease, NAFLD / MASLD, NASH / MASH, cholestatic disease, or any combination thereof. In some embodiments, the composition is administered orally, rectally, intravenously, intranasally, ocularly, subcutaneously, transdermally, or transmucosally.

[0043] In some embodiments, the invention provides a method for increasing bile acid deconjugation, wherein the method comprises administering the composition comprising the modified BSH.

[0044] In some embodiments, the method targets a specific species of BAs.

[0045] In some embodiments, the method increases deconjugation of circulating bile acids.

[0046] In some embodiment, the invention provides a pharmaceutical composition comprising a modified bile salt hydrolase (BSH) enzyme, formulated in a capsule or pill or any other delivery method, wherein the composition is effective to modulate bile acid metabolism in a subject.

[0047] In some embodiments, the modified BSH is administered at a concentration of about 0.1% to 100% by weight of the composition.

[0048] In some embodiments, the invention provides a composition comprising a genetically modified Bacteroides thetaiotaomicron and at least one carrier, wherein the genetically modified Bacteroides thetaiotaomicron expresses one or more heterologous genes encoding at least one bile salt metabolism enzyme; and wherein the bile salt metabolism enzyme encoded by the one or more heterologous genes is characterized by having enhanced enzymatic activity, lower or lack of activity, and / or substrate specificity and / or stability.

[0049] In some embodiments, the genetically modified Bacteroides thetaiotaomicron bears a knockout mutation in at least one of the genes encoding bile acid or bile salt metabolism enzyme.

[0050] In some embodiments, the at least one bile salt metabolism enzyme is a bile salt hydrolase (BSH).

[0051] In some embodiments, the BSH is a native Christensenella minuta BSH.

[0052] In some embodiments, the at least one bile salt metabolism enzyme comprises an amino acid sequence set forth as SEQ ID NO: 34, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33. In some embodiments, the at least one bile salt metabolism enzyme comprises an amino acid sequence set forth as SEQ ID NO: 7, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 4, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0053] In some embodiments, the genetically modified Bacteroides thetaiotaomicron expresses a combination of two or more heterologous genes encoding two or more bile salt metabolism enzymes, and wherein the two or more bile salt metabolism enzymes are characterized by having enhanced enzymatic activity, lower or lack of activity, and / or substrate specificity and / or stability, and, wherein the two or more bile salt metabolism enzymes, independently of each other, are characterized by having an amino acid sequence set forth as SEQ ID NO: 34, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, EQ ID NO: 7, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 4, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0054] In some embodiments, wherein the BSH comprises at least one amino acid substitution at position L67, K195, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 31 or 33.

[0055] In some embodiments, the BSH comprises a combination of at least two amino acid substitutions at any of the positions L67, KI 95, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 31 or 33.

[0056] In some embodiments, the BSH comprises a combination of multiple amino acid substitutions at any of the positions L67, KI 95, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 31 or 33.

[0057] In some embodiments, the BSH comprises at least one amino acid substitution at position Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 32.

[0058] In some embodiments, the BSH comprises at least one amino acid substitution at position Y34, H47, 157, L127, KI 89, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34. In some embodiments, the BSH comprises a combination of at least two amino acid substitutions at any of the positions Y34, H47, 157, L127, KI 89, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34.

[0059] In some embodiments, the BSH comprises a combination of multiple amino acid substitutions at any of the positions Y34, H47, 157, L127, KI 89, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34.

[0060] In some embodiments, the one or more heterologous genes encoding at least one bile salt metabolism enzyme comprise a polynucleotide sequence having at least 80% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 16 to SEQ ID NO:30 or SEQ ID NO: 51 to SEQ ID NO: 55.

[0061] In some embodiments, the one or more heterologous genes encoding at least one bile salt metabolism enzyme comprise a polynucleotide sequence having at least 90% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 16 to SEQ ID NO:30 or SEQ ID NO: 5 I to SEQ ID NO:55.

[0062] In some embodiments, the one or more heterologous genes encoding at least one bile salt metabolism enzyme comprise a polynucleotide sequence having between 80% to 99% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 16 to SEQ ID NO:30 or SEQ ID NO: 51 to SEQ ID NO: 55.

[0063] In some embodiments, the one or more heterologous genes encoding at least one bile salt metabolism enzyme comprise a polynucleotide sequence having at least 90% sequence identity to the polynucleotide sequence set forth SEQ ID NO: 41 to 43.

[0064] In some embodiments, the one or more heterologous genes encoding at least one bile salt metabolism enzyme comprise polynucleotide sequence having at least 90% sequence identity to the polynucleotide sequence set forth SEQ ID NO: 44.

[0065] In some embodiments, the genetically modified Bacteroides thetaiotaomicron further expresses one or more heterologous genes encoding bile acid acyl synthetase for succinyl (BAS-suc); and wherein the BAS-suc is encoded by the one or more heterologous genes comprising an amino acid sequence set forth as SEQ ID NO: 35 or SEQ ID NO: 50. In some embodiments, the one or more heterologous genes encoding bile acid acyl synthetase for succinyl (BAS-suc) has at least 90% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 45.

[0066] In some embodiments, the genetically modified Bacteroides thetaiotaomicron further expresses a BAS-Suc comprising an amino acid sequence set forth as SEQ ID NO: 50, a BSH having an amino acid sequence set forth as SEQ ID NO: 36, or a combination thereof.

[0067] In some embodiments, the one or more heterologous genes encoding BAS-Suc, BSH, or a combination thereof has at least between 60% to 100% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 46.

[0068] In some embodiments, the composition is formulated as an agent selected from the group consisting of capsule, pill, yogurt, drink, powder, tablet, suspension, aqueous solution, emulsion, syrup, cream, paste, gel, suppository, ointment, spray, foam, mill, and colloid.

[0069] In some embodiments, the composition is a pharmaceutical composition, and the carrier is pharmaceutically acceptable carrier.

[0070] In some embodiments, the concentration of the genetically modified Bacteroides thetaiotaomicron is from about 0.1% to about 100% by weight of the composition.

[0071] In some embodiments, the composition is for use as a medicament.

[0072] In some embodiments, for use in the treatment of a disease or condition associated with impaired metabolism and / or lipid metabolism.

[0073] In some embodiments, the disease or condition associated with impaired metabolism is selected from the group consisting of a metabolic disorder, metabolic syndrome, cardiovascular disease, cholestatic disease, hyperlipidemia, dyslipidemia, insulin resistance, type II diabetes, impaired glucose tolerance, obesity, liver disease, NAFLD / MASLD, NASH / MASH, cholestatic disease, or any combination thereof.

[0074] In some embodiments, the composition is a nutraceutical, supplement, medical food, and / or GRAS designated product.

[0075] In some embodiments, the invention provides a capsule comprising the composition the genetically modified Bacteroides thetaiotaomicron, wherein the genetically modified Bacteroides thetaiotaomicron is administered at a concentration of about 1 x 103CFU / animal (or below) to IxlO13CFU / animal (or higher).

[0076] In some embodiments, the invention provides a method of manufacture of a composition comprising a genetically modified bacterial cell and at least one carrier, wherein the genetically modified bacterial cell expresses one or more heterologous genes encoding at least one bile salt metabolism enzyme, wherein the method comprises the steps of: a) Generating a genetically modified bacterial strain, b) Transforming the genetically modified bacterial strain with at least one vector containing one or more heterologous genes encoding at least one bile salt metabolism enzyme; c) Expressing the at least one bile salt metabolism enzyme in the genetically modified bacterial strain; and, d) Preparing the composition comprising the genetically modified bacterial strain and at least one carrier.

[0077] BRIEF DESCRIPTION OF THE DRAWINGS

[0078] The following detailed description of various embodiments of the invention will be beter understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, illustrative embodiments are shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0079] Figure 1 depicts a representative schematic timeline and summary of a disease model experiment.

[0080] Figure 2 depicts a representative schematic timeline and summary of a disease model experiment.

[0081] Figure 3 depicts a representative exemplary enzymatic reaction for taurine formation with the disclosed BSH enzymes.

[0082] Figure 4 depicts a representative exemplary reaction for glycine formation with the disclosed BSH enzymes.

[0083] Figure 5 depicts representative data demonstrating an SDS-PAGE gel stained with Coomassie Blue to visualize total production of BSH proteins. Figure 6 depicts representative data of LCMS. Percent remaining of bile acids after 4, 8, 24, and 48 hour growth with Bacteroides thetaiotaomicron expressing BSH genes. n=l cultures per strain.

[0084] Figure 7A through Figure 7D depict representative data of EIC. Liquid chromatograms of Glycocholic acid (Figure 7A), Taurocholic acid (Figure 7B), Glycochenodeoxycholic acid (Figure 7C), and Taurochenodeoxycholic acid (Figure 7D). Data comprises of 4, 8, 24, and 48 hours.

[0085] Figure 8 depicts representative data of LCMS. Percent remaining of bile acids after 4, 8 and 24 hour growth with Bacteroides thetaiotaomicron expressing BSH genes. n=l cultures per strain.

[0086] Figure 9A through Figure 9D depict representative data of EIC. Liquid chromatograms of Glycocholic acid (Figure 9A), Taurocholic acid (Figure 9B), Glycochenodeoxycholic acid (Figure 9C), and Taurochenodeoxycholic acid (Figure 9D). Data comprises of 4, 8, and 24 hours.

[0087] Figure 10 depicts representative data of variant QC.

[0088] Figure 11 depicts representative data of variant QC ISTD.

[0089] Figure 12 depicts representative bile acid degradation assays performed with the native wild-type BSH enzymes. Percent remaining of bile acids after 4, 8 and 24 hour growth with Bacteroides thetaiotaomicron expressing BSH genes. Bacteroides thetaiotaomicron expressing native wild-type BSH enzymes are compared to the Bacteroides thetaiotaomicron control (VPI 5482). n=l cultures per strain.

[0090] Figure 13 depicts representative bile acid degradation assays performed with the BSH1 native enzyme and BSH1 L67Y variant. Percent remaining of bile acids after 4, 8 and 24 hour growth with Bacteroides thetaiotaomicron expressing BSH1 native enzyme or L67Y variant. Bacteroides thetaiotaomicron expressing BSH1 native enzyme or L67Y enzyme is compared to the Bacteroides thetaiotaomicron control (VPI 5482). n=l cultures per strain.

[0091] Figure 14 depicts representative bile acid degradation assays performed with the BSH2 variants. Percent remaining of bile acids after 4, 8 and 24 hour growth with Bacteroides thetaiotaomicron expressing BSH2 variants. Bacteroides thetaiotaomicron expressing BSH2 enzyme is compared to the Bacteroides thetaiotaomicron control (VPI 5482). n=l cultures per strain.

[0092] Figure 15 depicts representative bile acid degradation assays performed with the BSH1 variants. Percent remaining of bile acids after 4, 8 and 24 hour growth with Bacteroides thetaiotaomicron expressing BSH1 variants. Bacteroides thetaiotaomicron expressing BSH1 enzymes are compared to the Bacteroides thetaiotaomicron control (VPI 5482). n=l cultures per strain.

[0093] Figure 16 depicts representative bile acid degradation assays performed with the BSH1 IE2 variants. Percent remaining of bile acids after 4, 8 and 24 hour growth with Bacteroides thetaiotaomicron expressing BSH1 IE2 variants. Bacteroides thetaiotaomicron expressing BSH1 IE2 enzymes are compared to the Bacteroides thetaiotaomicron control (VPI 5482). n=l cultures per strain.

[0094] DETAILED DESCRIPTION

[0095] The present invention is now described more fully hereinafter with reference to the accompanying examples, in which embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art. According to some embodiments, the invention provides compositions and methods comprising at least one modified bile salt metabolism enzyme or a genetically modified Bacteroides thetaiotaomicron expressing one or more heterologous genes encoding at least one bile salt metabolism enzyme. The invention described herein provides a platform technology which has the precise capability to fine-tune bile acid metabolism with substrate specificity and novel activity levels and stability levels, not found in nature, specifically targeting the enterohepatic bile acid circulation.

[0096] Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0097] As used herein, each of the following terms has the meaning associated with it in this section.

[0098] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0099] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0100] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an RNA (e.g., mRNA), to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of RNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the RNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0101] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non- viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

[0102] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), RNA, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0103] “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared multiplied by 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

[0104] As used herein, a nucleotide sequence is “substantially homologous” or “substantially identical” to any of the nucleotide sequences described herein when its nucleotide sequence has a degree of identity with respect to the original nucleotide sequence at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 85%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%.

[0105] As used herein, an amino acid sequence is “substantially homologous” or “substantially identical” to any of the amino acid sequences described herein when its amino acid sequence has a degree of identity with respect to the original amino acid sequence of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 85%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%. The identity between two sequences can be determined by using the BLASTP algorithm for amino acid sequences or the BLASTN algorithm for nucleotide sequences (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)).

[0106] The term “variant” as used herein with respect to a nucleic acid refers (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequence substantially identical thereto. A variant may be a nucleic acid sequence that is substantially identical over the full length of the full gene sequence or a fragment thereof. The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the gene sequence or a fragment thereof.

[0107] The term “variant” as used with respect to a peptide or polypeptide refers to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Variant may also refer to a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. (Kyte et al., 1982, J. Mol. Biol. 157: 105- 132). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, incorporated fully herein by reference. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, as is understood in the art. Substitutions may be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties. A variant may be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or fragment thereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the amino acid sequence or a fragment thereof.

[0108] “Fragment” as used herein, refers to a nucleic acid sequence or amino acid sequence. In one embodiment, the fragment is a nucleic acid sequence that encodes a fragment of a protein. The fragment may be a fragment of a protein which retains its biologic activity. “Fragment” may also mean a fragment of a nucleic acid molecule. The fragments can be DNA fragments of the various nucleotide sequences that encode protein fragments. The fragments can be DNA fragments of DNA sequences having homology to at least one of the various nucleotide sequences that encode protein fragments set forth below. A fragment of a protein or nucleic acid may be 100% identical to the full length except missing at least one amino acid / nucleic acid from the N and / or C terminal, in each case with or without signal peptides and / or a methionine at position 1. Fragments may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more percent of the length of the particular full-length protein or nucleic acid, excluding any heterologous signal peptide added. The fragment may comprise a fragment of a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identical to the protein or nucleic acid and additionally comprise an N terminal methionine or heterologous signal peptide which is not included when calculating percent identity. Fragments may further comprise an N terminal methionine and / or a signal peptide.

[0109] A fragment of a nucleic acid sequence may be 100% identical to the full length except missing at least one nucleotide from the 5' and / or 3' end. When the nucleic acid sequence encodes a protein, the fragment of the nucleic acid sequence may be, in each case with or without sequences encoding signal peptides and / or a methionine at position 1. Fragments may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more percent of the length of the particular full length coding sequence, excluding any heterologous signal peptide added. The fragment may comprise a fragment that encode a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identical to the antibody and additionally optionally comprise sequence encoding an N terminal methionine or heterologous signal peptide which is not included when calculating percent identity. Fragments may further comprise coding sequences for an N terminal methionine and / or a signal peptide. The coding sequence encoding the N terminal methionine and / or signal peptide may be linked to a fragment of coding sequence.

[0110] “Isolated” as used herein means (1) altered or removed from the natural state and / or (2) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting) and / or otherwise previously associated, and / or (3) designed, produced, prepared, and / or manufactured by the hand of man. In some embodiments, a nucleic acid or a peptide naturally present in a living subject is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0111] In the context of the present disclosure, the following abbreviations for the commonly occurring nucleosides (nucleobase bound to ribose or deoxyribose sugar via N-glycosidic linkage) are used. “A” refers to adenosine, “C” refers to cytidine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0112] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNAmay include introns. The phrase nucleotide sequence that encodes a protein or an RNAmay also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s). In addition, the nucleotide sequence may contain modified nucleosides that are capable of being translated by translational machinery in a cell.

[0113] The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA or RNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0114] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

[0115] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0116] The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. By way of one non-limiting example, a promoter that is recognized by bacteriophage RNA polymerase and is used to generate the RNA by in vitro transcription.

[0117] By the term “modulating,” as used herein, means mediating a detectable increase or decrease in the level of expression of a gene or protein in a modified cell compared with an unmodified cell.

[0118] The term “endogenous,” as used herein, means originating from the same organism. For example, a protein that is endogenous to a cell is one that is encoded by a gene in the cell’s genome. In contrast, the term “heterologous” as used herein, means originating from a different organism.

[0119] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0120] The phrase “under transcriptional control” or “operatively linked” with reference to a promoter as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0121] As used herein, the term “knockout mutation” refers, without limitation, to targeted removal or inactivation of a specific gene and / or a portion of a gene, and / or multiple genes within an organism's genome. The term “knockout mutation” can be referred to as and interchangeable with the terms “gene knockout”, “gene deletion”, “gene inactivation”, or any other commonly used term. The “knockout mutation” of the invention can be done by any technique of the state of the art.

[0122] The term “genetically modified” refers, without limitation, to an organism whose genetic material has been artificially altered to produce a desired characteristic. Genetic modifications of the invention can include, without limitation, the introduction of new genes or enhancing, altering, or knocking out endogenous genes. In some genetic modifications, genes are transferred within the same species, and / or across species (creating transgenic organisms), and / or across kingdoms.

[0123] The term “mutated gene” refers, without limitation, to a change to a gene’s nucleotide sequence. In general, “mutated gene” can bear a silent mutation, namely mutations in DNA that do not have an observable effect on the organism's phenotype. In some instances, a “silent mutation” is a missense mutation, which change an amino acid to another amino acid, and nonsense mutations which change an amino acid to a stop codon, mutations in splice sites and insertions or deletions (indels).

[0124] As used herein, the term “heterologous gene” refers, without limitation to expression of a gene or a part thereof in a host organism that does not naturally have the gene or gene fragment.

[0125] The term “enzymatic activity” refers, without limitation, the rate of bile acid deconjugation or bile acid modification capability. In the context of the invention, the term “substrate specificity” refers, without limitation to selectivity or increased catalytic rate for a specific bile acid species (e.g. GCA) over other bile acid species. Ranges: throughout this disclosure, various aspects of the present disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0126] Description

[0127] The invention is based, in part, upon the surprising discovery that specific native and modified bile salt metabolism enzymes expressed in Bacteroides thetaiotaomicron increase and fine-tunes bile acid deconjugation. The present compositions and methods provide enzymes that are capable of deconjugating specific bile acids with increased precision. In some embodiments, the Bacteroides thetaiotaomicron are genetically modified. In some embodiments, the genetically modified Bacteroides thetaiotaomicron bears a knockout mutation in at least one of the genes encoding a bile acid or bile salt metabolism enzyme. In some embodiments, the genetically modified Bacteroides thetaiotaomicron expresses one or more heterologous genes encoding at least one native or modified bile salt metabolism enzyme. In some embodiments, the at least one bile salt metabolism enzyme is bile salt hydrolase (BSH).

[0128] The described compositions and methods are aimed at generating an observable effect on the phenotype in the form of expressing enzymes having better activity and / or improved substrate specificity. In some embodiments, the genetically modified Bacteroides thetaiotaomicron expresses one or more heterologous genes encoding at least one native bile salt metabolism enzyme. In some embodiments, the one or more heterologous genes encoding at least one bile salt metabolism enzyme comprise single or multiple mutations. In some embodiments, the modified bile salt metabolism enzyme encoded by the one or more mutated heterologous genes is characterized by having an enhanced enzymatic activity, lowered enzymatic activity, enhanced substrate specificity and / or enhanced stability.

[0129] Compositions

[0130] The invention relates, in part, to a composition comprising a bile salt metabolism enzyme. In some embodiments, the bile metabolism enzyme has enhanced enzymatic activity, lowered enzymatic activity, enhanced substrate specificity and / or enhanced stability.

[0131] In another aspect, the invention relates, in part, to a composition comprising a genetically modified bacterial cell and at least one carrier. In some embodiments, the genetically modified bacterial cell bears a knockout mutation in at least one of the genes encoding a bile acid or bile salt metabolism enzyme. In some embodiments, the genetically modified bacterial cell expresses one or more heterologous genes encoding at least one bile salt metabolism enzyme.

[0132] Modified bile salt hydrolase enzymes

[0133] The invention provides, in part, a modified bile salt metabolism enzyme. As used herein, the term “modified” refers, without limitation, to a protein bearing a change and / or multiple changes in its amino acid residues and polypeptide chains. Modified enzymes of the invention may have change / s of the spatial structure and / or physicochemical properties of protein molecules and may influence the main functional properties of the enzymes such as activity and substrate specificity.

[0134] Exemplary bile salt metabolism enzymes include, but are not limited to, Bile salt hydrolase, BAS-suc, 3-alpha hydroxysteroid dehydrogenase, 3-beta hydroxysteroid dehydrogenase, 6-alpha hydroxysteroid dehydrogenase, 6-beta hydroxysteroid dehydrogenase, 7-alpha hydroxysteroid dehydrogenase, 7-alpha hydroxysteroid dehydrogenase, 7-alpha hydroxysteroid dehydrogenase, 7-beta hydroxy steroid dehydrogenase, 12-alpha hydroxy steroid dehydrogenase, 12-beta hydroxysteroid dehydrogenase, 5-alpha reductase, Bile acid coenzyme A ligase, Bile acid coenzyme A transferase, 3-oxocholoyl-CoA 4-desaturase, Bile acid 7alpha-dehydratase, and, 3 -dehydro-bile acid delta(4,6)-reductase. In some embodiments, the modified bile salt metabolism enzyme is bile salt hydrolase (BSH). Exemplary BSH enzymes include, but are not limited to, Turicibacter sanguinis BSH Group I MOL361 (SEQ ID NO: 31), Turicibacter sanguinis BSH Group II H121, Turicibacter sanguinis BSH Group III 1E2 (SEQ ID NO: 33), Turicibacter sanguinis BSH Group IV MOL361 (SEQ ID NO: 32), Turicibacter sanguinis BSH Group V MOL361, Turicibacter sanguinis BSH Group V H121, Turicibacter sanguinis BSH Group VII 1E2, Turicibacter sanguinis BSH Group VIII MOL361, Turicibac / er_spQQ \ 543345. \ . Christensenella minuta Xlal, Christensenella minuta DSM 33407, Christensenella minuta DSM 22607, and Christensenella minuta BSH2 (SEQ ID NO: 34).

[0135] In some embodiments, the modified bile salt hydrolase (BSH) has at least 80% homology to an amino acid sequence set forth as SEQ ID NO: 31 to SEQ ID NO: 34. In some embodiments, the modified bile salt hydrolase (BSH) has at least 90% homology to an amino acid sequence set forth as SEQ ID NO: 31 to SEQ ID NO: 34. According to some embodiments, the above modified bile salt hydrolase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to an amino acid sequence set forth as SEQ ID NO: 31 to SEQ ID NO: 34.

[0136] In some embodiments, the modified bile salt hydrolase (BSH) has at least one amino acid substitution at position L67, K195, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NOs: 31 or 33. In some embodiments, the at least one substitution comprises L67Y, K195P, R297S, or Q328P, relative to SEQ ID NOs: 31 or 33. In some embodiments, the modified bile salt hydrolase (BSH) has a combination of at least two amino acid substitutions at any of the positions L67, KI 95, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NOs: 31 or 33. In some embodiments, the at least two substitutions comprise L67Y, K195P, R297S, or Q328P, relative to SEQ ID NOs: 31 or 33. In some embodiments, the modified bile salt hydrolase (BSH) has a combination of multiple amino acid substitutions at any of the positions L67, K195, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NOs: 31 or 33. In some embodiments, the multiple substitutions comprise L67Y, K195P, R297S, or Q328P, relative to SEQ ID NOs: 31 or 33. In some embodiments, the modified bile salt hydrolase (BSH) has at least one amino acid substitution at position Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 32. In some embodiments, the at least one substitution comprises Q328P, relative to SEQ ID NO: 32.

[0137] In some embodiments, the modified bile salt hydrolase (BSH) has at least one amino acid substitution at position Y34, H47, 157, L127, KI 89, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34. In some embodiments, the at least one substitution comprises Y34K, H47N, I57V, L127I, K189P, N292S, or R323P, relative to SEQ ID NO: 34. In some embodiments, the modified bile salt hydrolase (BSH) has a combination of at least two amino acid substitutions at any of the positions Y34, H47, 157, L127, K189, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34. In some embodiments, the at least two substitutions comprise Y34K, H47N, I57V, L127I, K189P, N292S, or R323P, relative to SEQ ID NO: 34. In some embodiments, the above modified bile salt hydrolase (BSH) has a combination of at least two amino acid substitutions at any of the positions Y34, H47, 157, L127, KI 89, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34. In some embodiments, the multiple substitutions comprise Y34K, H47N, I57V, L127I, K189P, N292S, or R323P, relative to SEQ ID NO: 34.

[0138] In some embodiments, the modified bile salt hydrolase (BSH) comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to an amino acid sequence set forth as SEQ ID NOs: 1-15. In some embodiments, the bile salt metabolism enzyme of the invention comprises amino acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0139] In some embodiments, the modified bile salt hydrolase (BSH) is a Turicibacter bile salt hydrolase (BSH). In some embodiments, the modified bile salt hydrolase (BSH) is Turicibacter sanguinis bile salt hydrolase (BSH). In some embodiments, the modified Turicibacter sanguinis bile salt hydrolase (BSH) comprises an amino acid sequence comprising SEQ ID NOs: 31-33. In some embodiments, the modified bile salt hydrolase (BSH) is at least 80% identical to SEQ ID NOs: 31 or 33 and comprise one or more amino acid substitution at position L67, K195, R297, or Q328, relative to SEQ ID NOs: 31 or 33. In some embodiments, the one or more substitutions comprise L67Y, K195P, R297S, or Q328P, relative to SEQ ID NOs: 31 or 33.

[0140] In some embodiments, the modified bile salt hydrolase (BSH) is at least 80% identical to SEQ ID NO:32 and comprise at least one amino acid substitution at position Q328, relative to SEQ ID NO:32. In some embodiments, the at least one substitution comprise Q328P, relative to SEQ ID NO: 32.

[0141] In some embodiments, the modified bile salt hydrolase (BSH) comprises at least one amino acid substitution at position L67, KI 95, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NOs: 31 or 33. In some embodiments, the at least one substitution comprises L67Y, K195P, R297S, or Q328P, relative to SEQ ID NOs: 31 or 33. In some embodiments, the modified bile salt hydrolase (BSH) comprises a combination of at least two amino acid substitutions at any of the positions L67, K195, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NOs: 31 or 33. In some embodiments, the at least two substitutions comprise L67Y, K195P, R297S, or Q328P, relative to SEQ ID NOs: 31 or 33. In some embodiments, the modified bile salt hydrolase (BSH) comprises a combination of multiple amino acid substitutions at any of the positions L67, K195, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NOs: 31 or 33. In some embodiments, the multiple substitutions comprise L67Y, K195P, R297S, or Q328P, relative to SEQ ID NOs: 31 or 33.

[0142] In some embodiments, the modified bile salt hydrolase (BSH) comprises an amino acid set forth as SEQ ID NOs: 8-15. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 8 and wherein the modified enzyme has the substitution K195P, with regard to SEQ ID NOs: 31 or 33, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 9 and wherein the modified enzyme has the substitution R297S, with regard to SEQ ID NOs: 1 or 33, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 10 and wherein the modified enzyme has the substitution Q328P, with regard to SEQ ID NOss: 31-33, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 10. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 11 and wherein the modified enzyme has the substitutions R297S and Q328P, with regard to SEQ ID NOs: 3 lor 33, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 11 . In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 12 and wherein the modified enzyme has the substitution K195P, with regard to SEQ ID NOs: 31 or 33, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 12. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 13 and w'herein the modified enzyme has the substitution R297S, with regard to SEQ ID NOs: 31 or 33, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 13. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 14 and wherein the modified enzyme has the substitution Q328P, with regard to SEQ ID NOs: 31-33, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 14. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 15 and wherein the modified enzyme has the substitutions R297S and Q328P, with regard to SEQ ID NOs: 31 or 33, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 15.

[0143] In some embodiments, the modified bile salt hydrolase (BSH) is a Christensenella bile salt hydrolase (BSH). In some embodiments, the modified bile salt hydrolase (BSH) is Christensenella minuta bile salt hydrolase (BSH). In some embodiments, the modified Christensenella minuta bile salt hydrolase (BSH) comprises an amino acid sequence comprising SEQ ID NO: 34.

[0144] In some embodiments, the modified C hristensenella minuta bile salt hydrolase (BSH) is at least 80% identical to SEQ ID NO: 34 and comprise one or more amino acid substitution at position Y34, H47, 157, L127, K189, N292, or R323, when, relative to SEQ ID NO: 34. In some embodiments, the one or more substitutions comprise Y34K, H47N, I57V, L127I, K189P, N292S, or R323P, relative to SEQ ID NO: 34.

[0145] In some embodiments, the modified Christensenella minuta bile salt hydrolase (BSH) comprises at least one amino acid substitution at position Y34, H47, 157, L127, KI 89, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34. In some embodiments, the at least one substitution comprises Y34K, H47N, I57V, L127I, K189P, N292S, or R323P, relative to SEQ ID NO: 34. In some embodiments, the modified Christensenella minuta bile salt hydrolase (BSH) comprises a combination of at least two amino acid substitutions at any of the positions Y34, H47, 157, L127, KI 89, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34. In some embodiments, the at least two substitutions comprise Y34K, H47N, I57V, L127I, K189P, N292S, or R323P, relative to SEQ ID NO: 34. In some embodiments, the modified Christensenella minuta bile salt hydrolase (BSH) comprises a combination of multiple amino acid substitutions at any of the positions Y34, H47, 157, L127, K189, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34. In some embodiments, the multiple substitutions comprise Y34K, H47N, I57V, L127I, K189P, N292S, or R323P, relative to SEQ ID NO: 34.

[0146] In some embodiments, the modified Christensenella minuta bile salt hydrolase (BSH) comprises an amino acid sequence comprising SEQ ID NOs: 1-7. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 1 and wherein the modified enzyme has the substitution KI 89P, with regard to SEQ ID NO: 34, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 1 . In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 2 and wherein the modified enzyme has the substitution N292S, with regard to SEQ ID NO: 34, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 2. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 3 and wherein the modified enzyme has the substitution R323P, with regard to SEQ ID NO: 34, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 3. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 4 and wherein the modified enzyme has the substitutions Y34K, 157 V, LI 271, and N292S, with regard to SEQ ID NO: 34, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 4. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 5 and wherein the modified enzyme has the substitutions Y34N, H47N, L127I, and N292S, with regard to SEQ ID NO: 34, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 5. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 6 and wherein the modified enzyme has the substitutions N292S and R323P, with regard to SEQ ID NO: 34, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 6. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 7 and wherein the modified enzyme has the substitutions K189P, N292S, and R323P, with regard to SEQ ID NO: 34, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 7.

[0147] Modified Bacterial Cell

[0148] In some embodiments, the invention relates to a genetically modified bacterial cell, wherein the genetically modified bacterial cell expresses one or more heterologous genes encoding at least one bile salt metabolism enzyme.

[0149] In some embodiments, the genetically modified bacterial cell bears a knockout mutation in at least one of the genes encoding a bile acid or bile salt metabolism enzyme. In some embodiments, the genetically modified bacterial cell bearing a knockout mutation in at least one of the genes encoding a bile acid or bile salt metabolism enzyme expresses one or more heterologous genes encoding at least one bile salt metabolism enzyme. In some embodiments, the genetically modified bacterial cell bearing a knockout mutation in at least one of the genes encoding a bile acid or bile salt metabolism enzyme expresses one or more mutated heterologous genes encoding at least one bile salt metabolism enzyme.

[0150] In some embodiments, the bacterial cell is a gut microbiota bacterium. A non-limiting list of the bacterial species includes Acidaminococcus spp., Actinomyces spp., Akkermansia muciniphila, Allobaculum spp., Anaerococcus spp., Anaerostipes spp., Bacteroides spp., Bacteroides Other, Bacteroides thetaiotaomicron, Bacteroides acidifaciens, Bacteroides coprophilus, Bacteroides fragilis, Bacteroides ovatus, Bacteroides uniformis, Barnesiellaceae spp., Bifidobacterium adolescentis, Bifidobacterium Other, Bifidobacterium spp., Bilophila spp., Blautia obeum, Blautia producta, Blautia Other, Blautia spp., Bulleidia spp., Catenibacterium spp., Chrisenella spp., Citrobacter spp., Clostridiaceae spp., Clostridiales Other, Clostridiales spp., Clostridium perfringens, Clostridium spp., Clostridium Other, Collinsella aerofaciens, Collinsella spp., Collinsella stercoris, Coprococcus catus, Coprococcus spp., Coriobacteriaceae spp., Desulfovibrionaceae spp., Dialister spp., Dorea formicigenerans, Dorea spp., Dorea Other, Eggerthella lenta, Enterobacteriaceae Other, Enterobacteriaceae spp., Enterococcus spp., Erysipelotrichaceae spp., Eubacterium biforme, Eubacterium biforme, Eubacterium dolichum, Eubacterium spp., Faecalibacterium prausnitzii, Fusobacterium spp., Gemellaceae spp., Haemophilus parainfiuenzae, Haemophilus Other, Helicobacter spp., Helicobacter Lachnospiraceae Other, Lachnospiraceae spp., Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus zeae, Lactobacillus spp., Lactobacillaceae spp., Lactococcus spp., Leuconostocaceae spp., Megamonas spp., Megasphaera spp., Methanobrevibacter spp., Mitsuokella multacida, Mitsuokella spp., Mucispirillum schaedleri, Odoribacter spp., Oscillospira spp., Parabacteroides distasonis, Parabacteroides spp., Paraprevotella spp., Paraprevotellaceae spp., Parvimonas spp., Pediococcus spp., Pediococcus Other, Peptococcus spp., Peptoniphilus spp., Peptostreptococcus anaerobius, Peptostreptococcus Other, Phascolarctobacterium spp., Prevotella copri, Prevotella spp., Prevotella ster corea, Prevotellaceae, Proteus spp., Rikenellaceae spp., Roseburia faecis, Roseburia spp., Ruminococcaceae Other, Ruminococcaceae spp., Ruminococcus bromii, Ruminococcus gnavus, Ruminococcus spp., Ruminococcus Other, Ruminococcus torques, Slackia spp., S24-7 spp., SMB 53 spp., Streptococcus anginosus, Streptococcus luteciae, Streptococcus spp., Streptococcus Other, Sutterella spp., Turicibacter spp., UC Bulleidia, UC Enterobacteriaceae, UC Faecalibacterium, UC Parabacteroides, UC Pediococcus, Varibaculum spp., Veillonella spp., Sutterella, Turicibacter, UC Clostridiales, UC Erysipelotrichaceae, UC Ruminococcaceae, Veillonella arvula, Veillonella spp., Veillonella dispar, and Weissella. In some embodiments, the bacterial cell is a gram- negative, obligate anaerobic bacterium. In some embodiments, the bacterial cell is Bacteroides thetaiotaomicron.

[0151] In some embodiments, the composition comprises at least one modified bacterial cell expressing one or more heterologous genes encoding at least one bile salt metabolism enzyme. In some embodiments, the at least one modified bacteria cell is Bacteroides thetaiotaomicron. In some embodiments, the Bacteroides thetaiotaomicron expresses a native or modified BSH. In some embodiments, he Bacteroides thetaiotaomicron expresses a native BAS-suc. In some embodiments, the composition comprises Bacteroides thetaiotaomicron and at least one additional genetically modified bacterial cell. In some embodiments, the at least one additional genetically modified bacterial cell expresses one or more mutated heterologous genes encoding at least one bile salt metabolism enzyme. In some embodiments, the at least on additional genetically modified bacterial cell is Bacteroides uniformis. In some embodiments, the composition comprises a genetically modified Bacteroides thetaiotaomicron and a genetically modified Bacteroides uniformis. In some embodiments, the composition comprises Bacteroides thetaiotaomicron, wherein Bacteroides thetaiotaomicron expresses a native or modified BSH and Bacteroides uniformis, wherein Bacteroides uniformis expresses a native BAS-suc.

[0152] In some embodiments, the bacterial cell is genetically modified. In some embodiments, the genetic modification is a knockout mutation in at least one gene encoding a bile acid or bile salt metabolism enzyme. Knockout mutations can include, but are not limited to, deletions of coding sequences, insertional disruptions of target genes, promoter or regulatory region deletions, conditional knockout systems, or nonfunctional allele gene replacement.

[0153] In some embodiments, the knockout mutation is a mutation that decreases the expression of at least one gene encoding a bile acid or bile salt metabolism enzyme. In some embodiments, the at least one gene encoding a bile acid or bile salt metabolism enzyme is selected from the group consisting of Bile salt hydrolase, BAS-suc, 3-alpha hydroxysteroid dehydrogenase, 3-beta hydroxysteroid dehydrogenase, 6-alpha hydroxysteroid dehydrogenase, 6-beta hydroxysteroid dehydrogenase, 7-alpha hydroxysteroid dehydrogenase, 7-alpha hydroxysteroid dehydrogenase, 7-alpha hydroxysteroid dehydrogenase, 7-beta hydroxy steroid dehydrogenase, 12-alpha hydroxysteroid dehydrogenase, 12-beta hydroxysteroid dehydrogenase, 5-alpha reductase, Bile acid coenzyme A ligase, Bile acid coenzyme A transferase, 3-oxocholoyl- CoA 4-desaturase, Bile acid 7alpha-dehydratase, and, 3 -dehydro-bile acid delta(4,6)- reductase. In some embodiments, the bacterial cell bears a knockout mutation in multiple genes. In some embodiments, the bacterial cell bears a knockout mutation in 1 gene, 2 genes, 3 genes, 4 genes, 5 genes, 6 genes, 7 genes, 8 genes, 9 genes, or 10 genes.

[0154] In some embodiments, the bacterial cell bears a knockout mutation in at least one gene encoding a bile salt hydrolase (bsh) or hydroxysteroid dehydrogenase (hsdh). In some embodiments, the at least one gene encoding a bile salt hydrolase (bsh) or hydroxy steroid dehydrogenase (hsdh) is selected from the group consisting of bshA, bshB, hsdhA, or any combination thereof.

[0155] In some embodiments, the composition comprises a genetically modified Bacteroides thetaiotaomicron and at least one carrier. In some embodiments, the genetically modified Bacteroides thetaiotaomicron bears a knockout mutation in at least one gene encoding a bile acid or bile salt metabolism enzyme; wherein the at least one gene encoding a bile acid or bile salt metabolism enzyme is selected from the group consisting of: bshA, bshB, hsdhA, or any combination thereof.

[0156] In some embodiments, the genetically modified bacterial cell bears a knockout mutation in at least one of the genes encoding a bile acid or bile salt metabolism enzyme. In some embodiments, the genetically modified bacterial cell bearing a knockout mutation in at least one of the genes encoding a bile acid or bile salt metabolism enzyme expresses one or more heterologous genes encoding at least one bile salt metabolism enzyme.

[0157] In some embodiments, the invention relates to a genetically modified bacterial cell, wherein the genetically modified bacterial cell expresses one or more heterologous genes encoding at least one bile salt metabolism enzyme. In some embodiments, the genetically modified bacterial cell expresses one or more heterologous genes encoding bile salt hydrolase (BSH).

[0158] Exemplary BSH enzymes include, but are not limited to, Turicibacter sanguinis BSH Group I MOL361 (SEQ ID NO: 31), Turicibacter sanguinis BSH Group II H121, Turicibacter sanguinis BSH Group III 1E2 (SEQ ID NO: 33), Turicibacter sanguinis BSH Group IV MOL361 (SEQ ID NO: 32), Turicibacter sanguinis BSH Group V MOL361, Turicibacter sanguinis BSH Group V H121, Turicibacter sanguinis BSH Group VII 1E2, Turicibacter sanguinis BSH Group VIII MOL361,

[0159] Turicibacter _sp001543345:1, Christensenella minuta Xlal, Christensenella minuta DSM 33407, Christensenella minuta DSM 22607, and Christensenella minuta BSH2 (SEQ ID NO: 34).

[0160] In some embodiments, the genetically modified bacterial cell expresses one or more heterologous genes encoding native bile salt hydrolase (BSH). In some embodiments, the one or more heterologous genes encoding native bile salt hydrolase (BSH) encode an amino acid sequence comprising at least 80% homology to an amino acid sequence comprising SEQ ID NO: 34, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33. In some embodiments, the one or more heterologous genes encoding native bile salt hydrolase (BSH) encode an amino acid sequence comprising at least 90% homology to an amino acid sequence comprising SEQ ID NO: 34, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33. In some embodiments, the one or more heterologous genes encoding native bile salt hydrolase (BSH) encode an amino acid sequence comprising SEQ ID NO: 34, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33.

[0161] In some embodiments, the genetically modified bacterial cell expresses one or more mutated heterologous genes encoding bile salt hydrolase (BSH). In some embodiments, the one or more mutated heterologous genes encoding bile salt hydrolase (BSH) encode an amino acid sequence comprising SEQ ID NOs: 1-15. In some embodiments, the genetically modified bacterial cell expresses at least one bile salt metabolism enzyme, wherein the at least one bile salt metabolism enzyme comprises an amino acid sequence set forth as SEQ ID NO: 1-15. In some embodiments, the bile salt metabolism enzyme of the invention comprises amino acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15.

[0162] In some embodiments, the genetically modified bacterial cell expresses a combination of two or more mutated heterologous genes encoding two or more bile salt metabolism enzymes. In some embodiments, the each of the two or more bile salt metabolism enzymes may, independently of each other, have an amino acid sequence set forth as SEQ ID NOs: 1-15.

[0163] In some embodiments, the at least one bile salt metabolism enzyme is bile salt hydrolase (BSH). In some embodiments, the at least one bile salt metabolism enzyme is a Turicibacter bile salt hydrolase (BSH). In some embodiments, the at least one bile salt metabolism enzyme is a Turicibacter sanguinis bile salt hydrolase (BSH). In some embodiments, the genetically modified bacterial cell expresses one or more heterologous genes encoding native Turicibacter sanguinis bile salt hydrolase (BSH). In some embodiments, the one or more heterologous genes encoding native Turicibacter sanguinis bile salt hydrolase (BSH) encode an amino acid sequence comprising SEQ ID NOs: 31-33.

[0164] In some embodiments, the genetically modified bacterial cell expresses one or more modified Turicibacter sanguinis BSH enzymes. In some embodiments, the modified enzymes are at least 80% identical to SEQ ID NOs: 31 or 33 and comprise one or more amino acid substitution at position L67, K195, R297, or Q328, relative to SEQ ID NOs: 31 or 33. In some embodiments, the one or more substitutions comprise L67Y, K195P, R297S, or Q328P, relative to SEQ ID NOs: 31 or 33. In some embodiments, the modified enzymes are at least 80% identical to SEQ ID NO: 32 and comprise one or more amino acid substitution at position Q328, relative to SEQ ID NO:32. In some embodiments, the one or more substitutions comprise Q328P, relative to SEQ ID NO: 32.

[0165] In some embodiments, the at least one bile salt metabolism enzyme is BSH comprising at least one amino acid substitution at position L67, K195, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NOs: 31 or 33. In some embodiments, the at least one substitution comprises L67Y, K195P, R297S, or Q328P, relative to SEQ ID NOs: 31 or 33. In some embodiments, the at least one bile salt metabolism enzyme is BSH comprising a combination of at least two amino acid substitutions at any of the positions L67, K195, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NOs: 31 or 33. In some embodiments, the at least two substitutions comprise L67Y, K195P, R297S, or Q328P, relative to SEQ ID NOs: 31 or 33. In some embodiments, the at least one bile salt metabolism enzyme is BSH comprising a combination of multiple amino acid substitutions at any of the positions L67, K195, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NOs: 31 or 33. In some embodiments, the multiple substitutions comprise L67Y, K195P, R297S, or Q328P, relative to SEQ ID NOs: 31 or 33.

[0166] In some embodiments, the genetically modified bacterial cell expresses one or more mutated heterologous genes encoding Turicibacter sanguinis bile salt hydrolase (BSH). In some embodiments, the one or more mutated heterologous genes encoding Turicibacter sanguinis bile salt hydrolase (BSH) encode an amino acid sequence comprising SEQ ID NOs: 8-15. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 8 and wherein the modified enzyme has the substitution K195P, with regard to SEQ ID NOs: 31 or 33, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 8 In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 9 and wherein the modified enzyme has the substitution R297S, with regard to SEQ ID NOs: 31 or 33, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 10 and wherein the modified enzyme has the substitution Q328P, with regard to SEQ ID NOss: 31-33, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 10. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 11 and wherein the modified enzyme has the substitutions R297S and Q328P, with regard to SEQ ID NOs: 31 or 33, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 11. In some1embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 12 and wherein the modified enzyme has the substitution K195P, with regard to SEQ ID NOs: 31 or 33, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 12. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 13 and wherein the modified enzyme has the substitution R297S, with regard to SEQ ID NOs: 31 or 33, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 13. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 14 and wherein the modified enzyme has the substitution Q328P, with regard to SEQ ID NOs: 1-33, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 14. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 15 and wherein the modified enzyme has the substitutions R297S and Q328R with regard to SEQ ID NOs: 31 or 33, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 15.

[0167] In some embodiments, the at least one bile salt metabolism enzyme is Christensenella bile salt hydrolase (BSH). In some embodiments, the at least one bile salt metabolism enzyme is Christensenella minuta bile salt hydrolase (BSH). In some embodiments, the genetically modified bacterial cell expresses one or more heterologous genes encoding native Christensenella minuta bile salt hydrolase (BSH). In some embodiments, the one or more heterologous genes encoding native Christensenella minuta bile salt hydrolase (BSH) encode an amino acid sequence comprising SEQ ID NO: 34.

[0168] In some embodiments, the genetically modified bacterial cell expresses one or more modified Christensenella minuta BSH enzymes. In some embodiments, the modified enzymes are at least 80% identical to SEQ ID NO: 34 and comprise one or more amino acid substitution at position Y34, H47, 157, L127, KI 89, N292, or R323, when, relative to SEQ ID NO: 34. In some embodiments, the one or more substitutions comprise Y34K, H47N, I57V, L127I, K189P, N292S, or R323P, relative to SEQ ID NO: 34.

[0169] In some embodiments, the at least one bile salt metabolism enzyme is BSH comprising at least one amino acid substitution at position Y34, H47, 157, L127, KI 89, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34. In some embodiments, the at least one substitution comprises Y34K, H47N, I57V, L127I, K189P, N292S, or R323P, relative to SEQ ID NO: 34. In some embodiments, the at least one bile salt metabolism enzyme is BSH comprising a combination of at least two amino acid substitutions at any of the positions Y34, H47, 157, L127, KI 89, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34. In some embodiments, the at least two substitutions comprise Y34K, H47N, I57V, L127I, K189P, N292S, or R323P, relative to SEQ ID NO: 34. In some embodiments, the at least one bile salt metabolism enzyme is BSH comprising a combination of multiple amino acid substitutions at any of the positions Y34, H47, 157, L127, K189, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34. In some embodiments, the multiple substitutions comprise Y34K, H47N, I57V, L127I, K189P, N292S, or R323P, relative to SEQ ID NO: 34.

[0170] In some embodiments, the genetically modified bacterial cell expresses one or more mutated heterologous genes encoding Christensenella minutci bile salt hydrolase (BSH). In some embodiments, the one or more mutated heterologous genes encoding Christensenella minuta bi\e salt hydrolase (BSH) encode an amino acid sequence comprising SEQ ID NOs: 1-7. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 1 and wherein the modified enzyme has the substitution KI 89P, with regard to SEQ ID NO: 34, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 1. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 2 and wherein the modified enzyme has the substitution N292S, with regard to SEQ ID NO: 34, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 2. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 3 and wherein the modified enzyme has the substitution R323P, with regard to SEQ ID NO: 34, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 3 In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 4 and wherein the modified enzyme has the substitutions Y34K, 157 V, LI 271, and N292S, with regard to SEQ ID NO: 34, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 4. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 5 and wherein the modified enzyme has the substitutions Y34N, H47N, LI 271, and N292S, with regard to SEQ ID NO: 34, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 5. In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 6 and wherein the modified enzyme has the substitutions N292S and R323P, with regard to SEQ ID NO: 34, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 6 In some embodiments, the modified enzyme comprises a sequence with at least 90% identity to SEQ ID NO: 7 and wherein the modified enzyme has the substitutions K189P, N292S, and R323P, with regard to SEQ ID NO: 34, optionally, wherein the modified enzyme comprises or consists of SEQ ID NO: 7.

[0171] In some embodiments, the genetically modified bacterial cell further expresses one or more heterologous genes encoding bile acid acyl synthetase for succinyl (BAS-suc). In one embodiment, the composition comprises a nucleic acid molecule comprising a nucleic acid sequence encoding bile acid acyl synthetase for succinyl (BAS- suc) at least 80% identical to SEQ ID NO: 35, SEQ ID NO: 50, or a homologue thereof. In some embodiments, the one or more heterologous genes encoding bile acid acyl synthetase for succinyl (BAS-suc) encode an amino acid sequence comprising SEQ ID NO: 35, or a homologue thereof. In some embodiments, the one or more heterologous genes encoding bile acid acyl synthetase for succinyl (BAS-suc) encode an amino acid sequence comprising SEQ ID NO: 50, or a homologue thereof.

[0172] In some embodiments, the genetically modified bacterial cell expresses one or more heterologous genes encoding a BAS-Suc having an amino acid sequence set forth as SEQ ID NO: 50 and a BSH having an amino acid sequence set forth as SEQ ID No: 36. In some embodiments, the BAS-Suc and BSH are expressed co-transcriptionally.

[0173] In some embodiments, the genetically modified bacterial cell is Bacteroides thetaiotaomicron. In some embodiments, the Bacteroides thetaiotaomicron comprises a knockout mutation in at least one gene encoding a bile acid or bile salt metabolism enzyme. In some embodiments, the at least one gene encoding a bile acid or bile salt metabolism enzyme is selected from the group consisting of: bshA, bshB, hsdhA, or any combination thereof.

[0174] Polynucleotides

[0175] In another aspect, the invention relates, in part, to polynucleotides encoding a modified bile salt hydrolase (BSH). In some embodiments, the invention provides an isolated oligonucleotide having at least 85% sequence identity to the polynucleotide sequence set forth as SEQ ID NOs: 41-44. In some embodiments, the isolated oligonucleotide has at least 80% sequence identity to the polynucleotide sequence set forth as SEQ ID NOs: 41 -44. In some embodiments, the isolated oligonucleotide has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence set forth as SEQ ID NOs: 41-44. In some embodiments, the invention provides an isolated oligonucleotide having at least 85% sequence identity to the polynucleotide sequence set forth as SEQ ID NOs: 16-30 or 51-55. In some embodiments, the isolated oligonucleotide has at least 80% sequence identity to the polynucleotide sequence set forth as SEQ ID NOs: 16-30 or 51-55. In some embodiments, the isolated oligonucleotide has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence set forth as SEQ ID NOs: 16-30 or 51- 55. In some embodiments, the invention provides an isolated oligonucleotide having polynucleotide sequence set forth as SEQ ID NOs: 16-30 or 51-55.

[0176] In some embodiments, the invention provides an isolated oligonucleotide encoding the modified BSH according to one or more of the above embodiments. In some embodiments, the polynucleotides comprise a nucleic acid sequence encoding a modified bile salt hydrolase (BSH) enzyme at least 80% identical to SEQ ID NOs: 31 or 33 and comprising one or more amino acid substitution at position L67, K195, R297, or Q328, relative to SEQ ID NOs: 31 or 33. In some embodiments, the one or more substitutions comprise L67Y, K195P, R297S, or Q328P, relative to SEQ ID NOs: 31 or 33. In some embodiments, the polynucleotides comprise a nucleic acid sequence encoding a modified bile salt hydrolase (BSH) enzyme at least 80% identical to SEQ ID NO: 32 and comprising at least one amino acid substitution at position Q328, relative to SEQ ID NO:32. In some embodiments, the at least one substitution comprises Q328P, relative to SEQ ID NO: 32. In some embodiments, the polynucleotides comprise a nucleic acid sequence encoding a modified bile salt hydrolase (BSH) enzyme at least 80% identical to SEQ ID NO: 34 and comprising one or more amino acid substitution at position Y34, H47, 157, L127, KI 89, N292, or R323, relative to SEQ ID NO: 34. In some embodiments, the one or more substitutions comprise Y34K, H47N, I57V, L127I, K189P, N292S, or R323P, relative to SEQ ID NO: 34.

[0177] In some embodiments, the present invention provides a composition comprising a nucleic acid molecule encoding the modified BSH according to one or more of the above embodiments. In some embodiments, the composition comprises a nucleic acid molecule comprising a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence set forth as SEQ ID NOs: 41-44. In some embodiments, the composition comprises a nucleic acid molecule comprising a nucleic acid sequence encoding a modified bile salt hydrolase (BSH) enzyme at least 90% identical to SEQ ID NOs: 31 or 33 and comprising one or more amino acid substitution at position L67, K195, R297, or Q328, relative to SEQ ID NOs: 31 or 33. In some embodiments, the one or more substitutions comprise L67Y, K195P, R297S, or Q328P, relative to SEQ ID NOs: 31 or 33. In some embodiments, the composition comprises a nucleic acid molecule comprising a nucleic acid sequence encoding a modified bile salt hydrolase (BSH) enzyme at least 90% identical to SEQ ID NO: 32 and comprising at least one amino acid substitution at position Q328, relative to SEQ ID NO:32. In some embodiments, the at least one substitution comprises Q328P, relative to SEQ ID NO: 32. In some embodiments, the composition comprises a nucleic acid molecule comprising a nucleic acid sequence encoding a modified bile salt hydrolase (BSH) enzyme at least 90% identical to SEQ ID NO: 34 and comprising one or more amino acid substitution at position Y34, H47, 157, L127, KI 89, N292, or R323, relative to SEQ ID NO: 34. In some embodiments, the one or more substitutions comprise Y34K, H47N, I57V, L127I, K189P, N292S, or R323P, relative to SEQ ID NO: 34. In some embodiments, the composition comprises a nucleic acid molecule comprising a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence set forth as SEQ ID NOs: 16-30 or 51-55. In some embodiments, the composition comprises a nucleic acid molecule comprising a nucleic acid sequence encoding bile acid acyl synthetase for succinyl (BAS-suc). In one embodiment, the composition comprises a nucleic acid molecule comprising a nucleic acid sequence encoding bile acid acyl synthetase for succinyl (BAS-suc) at least 80% identical to SEQ ID NO: 35 or SEQ ID NO: 50. In some embodiments, the composition comprises a nucleic acid molecule comprising a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 45 or SEQ ID NO: 46.

[0178] In some embodiments, the genetically modified bacterial cell expresses one or more heterologous genes encoding bile salt hydrolase (BSH). In some embodiments, the one or more heterologous genes encoding bile salt hydrolase (BSH) comprise a nucleic acid sequence comprising SEQ ID NOs: 41-44.

[0179] In some embodiments, the one or more mutated heterologous genes encoding at least one bile salt metabolism enzyme have polynucleotide sequence having at least 90% sequence identity to the polynucleotide sequence set forth as SEQ ID NOs: 41 to 43. In some embodiments, the one or more mutated heterologous genes encoding at least one Turicibacter sanguinis bile salt metabolism enzyme have polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence set forth as SEQ ID NOs: 41 to 43.

[0180] In some embodiments, the one or more mutated heterologous genes encoding at least one bile salt metabolism enzyme have polynucleotide sequence having at least 80% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 44. In some embodiments, the one or more mutated heterologous genes encoding at least one Christensenella nrinuta bile salt metabolism enzyme have polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence set forth as SEQ ID NO:44.

[0181] In some embodiments, the genetically modified bacterial cell expresses one or more mutated heterologous genes encoding bile salt hydrolase (BSH). In some embodiments, the one or more mutated heterologous genes encoding bile salt hydrolase (BSH) comprise a nucleic acid sequence comprising SEQ ID NOs: 16-30 or 51-55.

[0182] In some embodiments, the one or more mutated heterologous genes encoding at least one bile salt metabolism enzyme have polynucleotide sequence having at least 80% sequence identity to the polynucleotide sequence set forth as SEQ ID NOs: 16-30 or 51-55. In some embodiments, the one or more mutated heterologous genes encoding at least one bile salt metabolism enzyme have polynucleotide sequence having at least 80% sequence identity to the polynucleotide sequence set forth as SEQ ID NOs: 16-30. In some embodiments, the one or more mutated heterologous genes encoding at least one bile salt metabolism enzyme have polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence set forth as SEQ ID NOs: 16-30 or 51-55.

[0183] In some embodiments, the genetically modified bacterial cell expresses one or more mutated heterologous genes encoding a Turicibacter bile salt hydrolase (BSH). In some embodiments, the genetically modified bacterial cell expresses one or more mutated heterologous genes encoding Turicibacter sanguinis bile salt hydrolase (BSH). In some embodiments, the one or more mutated heterologous genes encoding Turicibacter sanguinis bile salt hydrolase (BSH) comprise a nucleic acid sequence comprising SEQ ID NOs: 23-30 or 51-54.

[0184] In some embodiments, the genetically modified bacterial cell expresses one or more mutated heterologous genes encoding a Christensenella bile salt hydrolase (BSH). In some embodiments, the genetically modified bacterial cell expresses one or more mutated heterologous genes encoding Christensenella minuta bile salt hydrolase (BSH). In some embodiments, the one or more mutated heterologous genes encoding Christensenella minuta bile salt hydrolase (BSH) comprise a nucleic acid sequence comprising SEQ ID NOs: 16-22 or 55.

[0185] In some embodiments, the genetically modified bacterial cell further expresses one or more heterologous genes encoding bile acid acyl synthetase for succinyl (BAS-suc). In some embodiments, the one or more heterologous genes encoding bile acid acyl synthetase for succinyl (BAS-suc) encode an amino acid sequence comprising SEQ ID NO: 35. In some embodiments, the one or more heterologous genes encoding bile acid acyl synthetase for succinyl (BAS-suc) encode an amino acid sequence comprising SEQ ID NO: 50. In some embodiments, the one or more heterologous genes encoding bile acid acyl synthetase for succinyl (BAS-suc) comprise a nucleic acid sequence having at least 80% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 45.

[0186] In some embodiments, the genetically modified bacterial cell expresses one or more heterologous genes encoding a BAS-Suc having an amino acid sequence set forth as SEQ ID NO: 50 and a BSH having an amino acid sequence set forth as SEQ ID NO: 36. In some embodiments, the one or more heterologous genes encoding BAS-Suc and BSH comprise a nucleic acid sequence having at least 80% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 46. In some embodiments, the BAS-Suc and BSH are expressed co-transcriptionally.

[0187] In some embodiments, the genetically modified bacterial cell is Bacteroides thetaiotaomicron. In some embodiments, the Bacteroides thetaiotaomicron comprises a knockout mutation in at least one gene encoding a bile acid or bile salt metabolism enzyme. In some embodiments, the at least one gene encoding a bile acid or bile salt metabolism enzyme is selected from the group consisting of: bshA, bshB, hsdhA, or any combination thereof.

[0188] In some embodiments, the cell comprises a nucleic acid molecule comprising a promotor and a sequence encoding a protein operatively linked to the promoter. In some embodiments, the nucleic acid molecule can be incorporated into a vector for delivery to a cell. In some embodiments, the nucleic acid molecule is integrated into the genome of the cell.

[0189] In some embodiments, the invention provides an expression vector comprising the isolated oligonucleotide according to one or more of the embodiments operably linked to at least one promoter.

[0190] In some embodiments, the invention provides a composition comprising at least one of: the modified bile salt hydrolase (BSH) according to one or more of the above embodiments, the isolated oligonucleotide according to one or more of the above embodiments, expression vector according to one or more of the above embodiments, or any combination thereof, and at least one carrier. In some embodiments, the composition is a pharmaceutical composition, and the carrier Is pharmaceutically acceptable carrier. In some embodiments, the composition is bacterial composition comprising at least one bacterial species comprising at least one the modified bile salt hydrolase (BSH) according to one or more of the above embodiments, the isolated oligonucleotide according to one or more of the above embodiments, expression vector according to one or more of the above embodiments, or any combination thereof.

[0191] In some embodiments, the invention provides a bacterial cell comprising at least one of: the modified bile salt hydrolase (BSH) according to one or more of the above embodiments, the isolated oligonucleotide according to one or more of the above embodiments, expression vector according to one or more of the above embodiments, or any combination thereof, and at least one carrier.

[0192] The nucleic acid molecule can be cloned into a number of types of vectors. For example, the nucleic acid molecule can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, and a cosmid. In some embodiments, the vectors are expression vectors. Numerous expression vector systems exist that comprise at least a part or all of the compositions discussed above. Prokaryote- and / or eukaryote-vector based systems can be employed to produce polynucleotides, or their cognate polypeptides. The vectors can be suitable for replication and integration. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0193] In some embodiments, the expression vectors or plasmids are suitable for use in any prokaryotic host cells. The vectors or plasmids disclosed herein may be employed for the expression of one or more heterologous or endogenous polypeptides, regulatory elements, or biosynthetic components, and may include one or more selectable marker genes, such as antibiotic resistance genes. Non-limiting examples of antibiotic resistance genes include ampicillin, kanamycin, chloramphenicol, tetracycline, spectinomycin, and streptomycin. In some embodiments, the vector or plasmid is an antibiotic resistance plasmid.

[0194] In some embodiments, the expression vectors or plasmids may include a kill-switch module system. Exemplary kill-switch module systems include, but are not limited to, an auxotrophic gene operably linked to a promoter or a toxin-antitoxin system such as ccdA / ccdB, hok / sok, or44yrrE / mazF, configured such that loss of the plasmid or presence / absence of an inducer results in activation of the toxin and subsequent cell death.

[0195] In some embodiments, the expression vectors or plasmids may include an auxotrophic system for colonization and engraftment. In some embodiments, the auxotrophic system introduces the described therapeutic bacterial cells into an environment, such as the gut, and ensures their containment.

[0196] In some embodiments, the expression vectors or plasmids provide a pass- through, transient approach for delivery of the described therapeutic bacterial cells. In some embodiments, the nucleic acid sequence is integrated into the genome of the cell. Therefore, in some embodiments, the vectors are integration vectors. An integration vector may comprise a nucleic acid sequence encoding a polypeptide described herein, a selectable marker, and a nucleic acid sequence homologous to a region of the genome of the cell to guide integration via homologous recombination.

[0197] In some embodiments, the nucleic acid molecule can be delivered to a cell directly, without the use of a vector. In some embodiments, the nucleic acid molecule comprises synthetic DNA. In some embodiments, the nucleic acid molecule is incorporated into the genome of a cell by any genome editing technique known in the art (e.g., CRISPR-Cas). In some embodiments, the nucleic acid molecule is integrated into the genome through endogenous homologous recombination or non-homologous end joining (NHEJ).

[0198] In some embodiments, the nucleic acid sequence encoding a polypeptide is under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. An enhancer may be one naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence. Such an enhancer can be referred to as “endogenous.” Alternatively, a recombinant or heterologous enhancer refers also to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not “naturally occurring,” e.g., containing different elements of different transcriptional regulatory regions, and / or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning, and / or nucleic acid amplification technology, including PCR, in connection with the compositions disclosed herein (e g., U.S. Patent 4,683,202, U.S. Patent 5,928,906).

[0199] Useful expression vectors include, but are not limited to, pACYDuet, pRSD GPD, trc, pD431, PD434, pD451, pD454 (DNA 2.0 (now ATUM)), pET3 (Novagen), pRSET (Thermo-Fisher), pBluescript vectors, Phagescript vectors, pNH8A, [rho]NH16a, pNH18A, [rho]NH46A (Stratagene Cloning Systems, Inc.); ptrc99a, pKK223-3, [rho]KK233-3, pDR540, pRIT5 (Pharmacia Bio-tech, Inc.); pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pACYC177, pACYC184, pRSFIOIO, pBW22 (Wilms et al., 2001, Biotechnology and Bioengineering, 73 (2) 95- 103), Yep, Yep, pYES2 (Invitrogen), pESC series (Stratagene / Agilent), pYES-DEST52 / pYES-DEST53 (Invitrogen), pRS series expression vectors, pCM190, pCM189, pTEF- based vectors, EasyClone System (Addgene), pFA6a-based expression cassettes, or variants thereof.

[0200] Useful integration vectors include, but are not limited to pINT-ts, pGRG36, pGRG25, pUC18-mini-Tn7T-Gm-Lacz, CRIM plasmids, X-Red recombineering vectors, pRS series integrative vectors (e.g., pRS306, pRS303), pFA6a series vectors, pIS series vecors, Yip351, Yip5, pSpin series plasmids and other CAST systems, or variants thereof. Further useful plasmids are well known to the person skilled in the art and are described e.g., in “Cloning Vectors” (Eds. Pouwels P H. et al. Elsevier, Amsterdam-New York-Oxford, 1985).

[0201] A nucleic acid molecule of the pr”sent’invention can be inserted into a vector by a variety of means known to the art. For example, design and construction of a vector can be achieved through complementary end PCR product ligation cloning, and homologous recombination. In some embodiments, a vector may be constructed using the Gibson assembly method (Gibson et al., Nat Methods, 2009, 6:343-345). Other suitable methods will be known to those skilled in the art.

[0202] In order to assess the expression of the polypeptides of the invention or portions thereof, the vector can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or transformed. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a cotransfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in host cells.

[0203] Suitable reporter genes may include, but are not limited to, genes encoding luciferase, beta-galactosidase, neo, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal ” flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[0204] Pharmaceutical Compositions

[0205] The invention provides various pharmaceutical compositions comprising a bile salt metabolism enzyme. In some embodiments, the invention relates to pharmaceutical compositions comprising a genetically modified bacterial cell and at least one carrier, wherein the modified bacterial cell expresses one or more heterologous genes encoding at least one bile salt metabolism enzyme. In some embodiments, the composition is a pharmaceutical composition, and the carrier is pharmaceutically acceptable carrier.

[0206] In various aspects, the composition comprises the modified bile salt metabolism enzyme or genetically modified bacterial cell expressing a bile salt metabolism enzyme and a one or more stabilizers. In various embodiments, the stabilizer to compound weight ratio is less than 50%. In one embodiment, the stabilizer comprises a biocompatible polymer. Examples of stabilizers include, but are not limited to, biocompatible polymer, a biodegradable polymer, a multifunctional linker, starch, modified starch, and starch derivatives, gums, including but not limited to polymers, polypeptides, albumin, amino acids, thiols, amines, carboxylic acid and combinations or derivatives thereof, citric acid, xanthan gum, alginic acid, other alginates, benitoniite, veegum, agar, guar, locust bean gum, gum47yrrolc, quince psyllium, flax seed, okra gum, arabinoglactin, pectin, tragacanth, scleroglucan, dextran, amylose, amylopectin, dextrin, etc., cross-linked polyvinylpyrrolidone, ion-exchange resins, potassium polymethacrylate, carrageenan (and derivatives), gum karaya and biosynthetic gum, polycarbonates (linear polyesters of carbonic acid); microporous materials (bisphenol, a microporous poly(vinylchloride), micro-porous polyamides, microporous modacrylic copolymers, microporous styrene-acrylic and its copolymers); porous polysulfones, halogenated poly(vinylidene), polychloroethers, acetal polymers, polyesters prepared by esterification of a dicarboxylic acid or anhydride with an alkylene polyol, poly(alkylenesulfides), phenolics, polyesters, asymmetric porous polymers, cross-linked olefin polymers, hydrophilic microporous homopolymers, copolymers or interpolymers having a reduced bulk density, and other similar materials, poly(urethane), cross-linked chain-extended poly(urethane), poly(imides), poly(benzimidazoles), collodion, regenerated proteins, semi-solid cross-linked polyvinylpyrrolidone), monomeric, dimeric, oligomeric or long-chain, copolymers, block polymers, block co-polymers, polymers, PEG, dextran, modified dextran, polyvinylalcohol, polyvinylpyrollidone, polyacrylates, polymethacrylates, polyanhydrides, polypeptides, albumin, alginates, amino acids, thiols, amines, carboxylic acids, or combinations thereof.

[0207] The compositions may be formulated in a pharmaceutically acceptable excipient, such as wetting agents, buffers, disintegrants, binders, fillers, flavoring agents and liquid carrier media such as sterile water, water / ethanol etc. The compositions should be suitable for administration either by topical administration or injection or inhalation or catheterization or instillation or transdermal introduction into any of the various body cavities including the alimentary canal, the vagina, the rectum, the bladder, the ureter, the urethra, the mouth, etc. For oral administration, the pH of the composition is preferably in the acid range (e.g., 2 to 7) and buffers or pH adjusting agents may be used. The contrast media may be formulated in conventional pharmaceutical administration forms, such as tablets, capsules, powders, solutions, dispersion, syrups, suppositories etc.

[0208] The compositions of the invention can be formulated and administered to a subject, as now described. The invention encompasses the preparation and use of pharmaceutical compositions comprising the compositions of the invention useful for the delivery of a therapeutic agent to a cell. The invention also encompasses the preparation and use of pharmaceutical compositions comprising the compositions of the invention useful for the treatment of a disease or disorder. The invention also encompasses the preparation and use of pharmaceutical compositions comprising the compositions of the invention useful for improved cell penetration.

[0209] Such a pharmaceutical composition may consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

[0210] In various embodiments, the pharmaceutical compositions useful in the methods of the invention may be administered, by way of example, systemically, parenterally, or topically, such as, in oral formulations, inhaled formulations, including solid or aerosol, and by topical or other similar formulations. In addition to the appropriate therapeutic composition, such pharmaceutical compositions may contain pharmaceutically acceptable carriers and other ingredients known to enhance and facilitate drug administration. Other possible formulations, such as nanoparticles, liposomes, resealed erythrocytes, and immunologically based systems may also be used to administer an appropriate modulator thereof, according to the methods of the invention.

[0211] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[0212] Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, ophthalmic, intrathecal and other known routes of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient (e.g., a modified bile salt metabolism enzyme and / or a genetically modified bacterial cell), and immunologically-based formulations.

[0213] A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0214] The relative amounts of the active ingredient (e.g., a modified bile salt metabolism enzyme and / or a genetically modified bacterial cell), the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) active ingredient. In various embodiments, the composition comprises at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% (w / w) active ingredient.

[0215] In some embodiments, the concentration of the genetically modified Bacteroides thetaiotaomicron is at least 5% by weight of the composition. In some embodiments, the concentration of the genetically modified Bacteroides thetaiotaomicron is from about 0.1% to 99.9% by weight of the composition. In one embodiment, the concentration of the genetically modified Bacteroides thetaiotaomicron is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% by weight of the composition.

[0216] In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents.

[0217] In some embodiments, the one or more additional pharmaceutically active agents is a serotonin receptor agonist. Exemplary serotonin receptor agonist can include, but is not limited to, 5-HT, psilocybin, psilocin, N,N-dimethyltryptamine, 5-methoxy- N,N-dimethyltryptamine, bufotenin, lysergic acid diethylamide, ergine (LSA), mescaline, 2,5-dimethoxy-4-bromophenethylamine, 251-NBOMe, 3,4-methylenedioxyamphetamine, or 2,5-dimethoxy-4-methylamphetamine.

[0218] In some embodiments, the pharmaceutical composition is formulated such that the composition exhibits chemical and physical stability within a pH range of approximately 5.0 to 7.0. In some embodiments, the pharmaceutical composition further comprises a buffering system to maintain the pH within the desired range. Exemplary buffering systems can include, but are not limited to, citrate, phosphate, or acetate buffer.

[0219] Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

[0220] A formulation of a pharmaceutical composition of the invention suitable for oral administration may be prepared, packaged, or sold in the form of a discrete solid dose unit including, but not limited to, a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the active ingredient. Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, or an emulsion. In some embodiments, the composition is a solid composition, a semisolid composition, or a liquid composition. A non-limiting list of the compositions of the invention includes powder, capsule, tablet, suspension, aqueous solution, emulsion, syrup, cream, paste, gel, suppository, ointment, spray, foam, mill, and colloid. As used herein, the term “semi-solid” refers, without limitation, to a state that is in between a solid and a liquid. Another name for a semi-solid is a quasi-solid. At the microscopic scale, it has a disordered structure unlike the more common solids.

[0221] A tablet comprising the active ingredient (e.g., a modified bile salt metabolism enzyme and / or a genetically modified bacterial cell) may, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients. Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycolate. Known surface active agents include, but are not limited to, sodium lauryl sulphate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, com starch and alginic acid. Known binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricating agents include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.

[0222] Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. By way of example, a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Further by way of example, tablets may be coated using methods described in U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotically-controlled release tablets. Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide pharmaceutically elegant and palatable preparation.

[0223] Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.

[0224] Soft gelatin capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such soft capsules comprise the active ingredient, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.

[0225] Liquid formulations of a pharmaceutical composition of the invention which are suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use. In some embodiments, the composition is for oral administration. In some embodiments, the composition is a discrete solid dose unit including, but not limited to, a hard or soft capsule, a tablet, a cachet, a troche, or a lozenge, each containing a predetermined amount of the modified bile salt metabolism enzyme and / or genetically modified bacterial cell.

[0226] Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, and hydroxypropylmethylcellulose. Known dispersing or weting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g. polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and acetyl alcohol.

[0227] Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

[0228] Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or weting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.

[0229] A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.

[0230] Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e., such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.

[0231] Parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of an individual and administration of the pharmaceutical composition through the breach in the tissue. Parental administration can be local, regional or systemic. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, intravenous, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrastemal injection, and intratumoral. In some embodiments, the composition is designed for oral, rectal, intravenous, intraperitoneal, transdermal, transmucosal, intranasal, ocular, or subcutaneous administration. Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0232] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0233] Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions. Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w / w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

[0234] A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, and preferably from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent / powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. Preferably, such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. More preferably, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. In some embodiments, dry powder compositions include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

[0235] Low boiling propellants generally include liquid propellants having a boiling point of below 65 °F at atmospheric pressure. Generally, the propellant may constitute 50 to 99.9% (w / w) of the composition, and the active ingredient may constitute 0.1 to 20% (w / w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (in some embodiments having a particle size of the same order as particles comprising the active ingredient).

[0236] Pharmaceutical compositions of the invention formulated for pulmonary delivery may also provide the active ingredient in the form of droplets of a solution or suspension. Such formulations may be prepared, packaged, or sold as aqueous or dilute alcoholic solutions or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration preferably have an average diameter in the range from about 0.1 to about 200 nanometers.

[0237] The formulations described herein as being useful for pulmonary delivery are also useful for intranasal delivery of a pharmaceutical composition of the invention.

[0238] Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers.

[0239] Such a formulation is administered in the manner in which snuff is taken i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nares. Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w / w) and as much as 100% (w / w) of the active ingredient, and may further comprise one or more of the additional ingredients described herein.

[0240] A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may, for example, contain 0.1 to 20% (w / w) active ingredient, the balance comprising an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder or an aerosolized or atomized solution or suspension comprising the active ingredient. Such powdered, aerosolized, or aerosolized formulations, when dispersed, preferably have an average particle or droplet size in the range from about 0.1 nanometers to about 2000 micrometers, and may further comprise one or more of the additional ingredients described herein.

[0241] A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1- 1.0% (w / w) solution or suspension of the active ingredient in an aqueous or oily liquid carrier. Such drops may further comprise buffering agents, salts, or one or more other of the additional ingredients described herein. Other opthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form or in a liposomal preparation.

[0242] As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or weting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed., 1985, Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.

[0243] Administration of the compounds of the present invention or the compositions thereof may be continuous or intermittent, depending, for example, upon the recipient’s physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of the agents of the invention may be essentially continuous over a preselected period of time or may be in a series of spaced doses. Both local and systemic administration is contemplated. The amount administered will vary depending on various factors including, but not limited to, the composition chosen, the particular disease, the weight, the physical condition, and the age of the mammal, and whether prevention or treatment is to be achieved. Such factors can be readily determined by the clinician employing animal models or other test systems which are well known to the art.

[0244] One or more suitable unit dosage forms having the therapeutic agent(s) of the invention, which, as discussed below, may optionally be formulated for sustained release (for example using microencapsulation, see WO 94 / 07529, and U.S. Pat. No. 4,962,091 the disclosures of which are incorporated by reference herein), can be administered by a variety of routes including parenteral, including by intravenous and intramuscular routes, as well as by direct injection into the diseased tissue. For example, the therapeutic agent may be directly injected into the muscle. The formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to pharmacy. Such methods may include the step of bringing into association the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.

[0245] When the therapeutic agents of the invention are prepared for administration, they are preferably combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form. The total active ingredients in such formulations include from 0.1 to 99.9% by weight of the formulation. A “pharmaceutically acceptable” is a carrier, diluent, excipient, and / or salt that is compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof. The active ingredient for administration may be present as a powder or as granules; as a solution, a suspension or an emulsion.

[0246] Pharmaceutical formulations containing the therapeutic agents of the invention can be prepared by procedures known in the art using well known and readily available ingredients. The therapeutic agents of the invention can also be formulated as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous or intravenous routes.

[0247] The pharmaceutical formulations of the therapeutic agents of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension.

[0248] Thus, the therapeutic agent may be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampules, pre-fdled syringes, small volume infusion containers or in multi-dose containers with an added preservative. The active ingredients may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0249] It will be appreciated that the unit content of active ingredient or ingredients contained in an individual aerosol dose of each dosage form need not in itself constitute an effective amount for treating the particular indication or disease since the necessary effective amount can be reached by administration of a plurality of dosage units. Moreover, the effective amount may be achieved using less than the dose in the dosage form, either individually, or in a series of administrations.

[0250] The pharmaceutical formulations of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are well-known in the art. Specific nonlimiting examples of the carriers and / or diluents that are useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline solutions, such as phosphate buffered saline solutions pH 7.0-8.0.

[0251] In general, water, suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration contain the active ingredient, suitable stabilizing agents and, if necessary, buffer substances. Antioxidizing agents such as sodium bisulfate, sodium sulfite or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium Ethylenediaminetetraacetic acid (EDTA). In addition, parenteral solutions can contain preservatives such as benzalkonium chloride, methyl or propyl-paraben and chlorobutanol. Suitable pharmaceutical carriers are described in Remington’s Pharmaceutical Sciences, a standard reference text in this field.

[0252] The active ingredients of the invention may be formulated to be suspended in a pharmaceutically acceptable composition suitable for use in mammals and in particular, in humans. Such formulations include the use of adjuvants such as muramyl dipeptide derivatives (MDP) or analogs that are described in U.S. Patent Nos. 4,082,735; 4,082,736; 4,101,536; 4,185,089; 4,235,771; and 4,406,890. Other adjuvants, which are useful, include alum (Pierce Chemical Co ), lipid A, trehalose dimycolate and dimethyldioctadecylammonium bromide (DDA), Freund’s adjuvant, and IL 12. Other components may include a polyoxypropylene-polyoxy ethylene block polymer (Pluronic®), a non-ionic surfactant, and a metabolizable oil such as squalene (U.S. Patent No. 4,606,918).

[0253] Additionally, standard pharmaceutical methods can be employed to control the duration of action. These are well known in the art and include control release preparations and can include appropriate macromolecules, for example polymers, polyesters, polyamino acids, polyvinyl, 62yrroli donee, ethylenevinylacetate, methyl cellulose, carboxymethyl cellulose or protamine sulfate. The concentration of macromolecules as well as the methods of incorporation can be adjusted in order to control release. Additionally, the agent can be incorporated into particles of polymeric materials such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylenevinylacetate copolymers. In addition to being incorporated, these agents can also be used to trap the compound in microcapsules.

[0254] Accordingly, the composition of the present invention may be delivered via various routes and to various sites in a mammal body to achieve a particular effect (see, e.g., Rosenfeld et al., 1991; Rosenfeld et al., 1991a; Jaffe et al., supra; Berkner, supra). One skilled in the art will recognize that although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route. In one embodiment, the composition described above is administered to the subject by subretinal injection. In other embodiments, the composition is administered by intravitreal injection. Other forms of administration that may be useful in the methods described herein include, but are not limited to, direct delivery to a desired organ (e.g., the eye), oral, inhalation, intranasal, intratracheal, intravenous, intramuscular, subcutaneous, intradermal, and other parental routes of administration. Additionally, routes of administration may be combined, if desired. In another embodiments, route of administration is subretinal injection or intravitreal injection.

[0255] The active ingredients of the present invention can be provided in unit dosage form wherein each dosage unit, e.g., a teaspoonful, tablet, solution, or suppository, contains a predetermined amount of the composition, alone or in appropriate combination with other active agents. The term “unit dosage form” as used herein refers to physically discrete units suitable as unitary dosages for human and mammal subjects, each unit containing a predetermined quantity of the compositions of the present invention, alone or in combination with other active agents, calculated in an amount sufficient to produce the desired effect, in association with a pharmaceutically acceptable diluent, carrier, or vehicle, where appropriate. The specifications for the unit dosage forms of the present invention depend on the particular effect to be achieved and the particular pharmacodynamics associated with the composition in the particular host.

[0256] The pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of at least about 1 ng / kg, at least about 5 ng / kg, at least about 10 ng / kg, at least about 25 ng / kg, at least about 50 ng / kg, at least about 100 ng / kg, at least about 500 ng / kg, at least about 1 pg / kg, at least about 5 pg / kg, at least about 10 pg / kg, at least about 25 pg / kg, at least about 50 pg / kg, at least about 100 pg / kg, at least about 500 pg / kg, at least about 1 mg / kg, at least about 5 mg / kg, at least about 10 mg / kg, at least about 25 mg / kg, at least about 50 mg / kg, at least about 100 mg / kg, at least about 200 mg / kg, at least about 300 mg / kg, at least about 400 mg / kg, and at least about 500 mg / kg of body weight of the subject.

[0257] In some embodiments, the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of no more than about 1 ng / kg, no more than about 5 ng / kg, no more than about 10 ng / kg, no more than about 25 ng / kg, no more than about 50 ng / kg, no more than about 100 ng / kg, no more than about 500 ng / kg, no more than about 1 pg / kg, no more than about 5 pg / kg, no more than about 10 pg / kg, no more than about 25 pg / kg, no more than about 50 pg / kg, no more than about 100 pg / kg, no more than about 500 pg / kg, no more than about 1 mg / kg, no more than about 5 mg / kg, no more than about 10 mg / kg, no more than about 25 mg / kg, no more than about 50 mg / kg, no more than about 100 mg / kg, no more than about 200 mg / kg, no more than about 300 mg / kg, no more than about 400 mg / kg, and no more than about 500 mg / kg of body weight of the subject. Also contemplated are dosage ranges between any of the doses disclosed herein. Typically, dosages which may be administered in a method of the invention to a subject, in some embodiments a human, range in amount from 0.5 pg to about 100 g per kilogram of body weight of the subject. While the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of subject and type of disease state being treated, the age of the subject and the route of administration. In some embodiments, the dosage of the compound will vary from about 1 pg to about 10 mg per kilogram of body weight of the subject. In other embodiments, the dosage will vary from about 3 pg to about 1 mg per kilogram of body weight of the subject.

[0258] The compositions may be administered to a subject as frequently as several times daily, or it may be administered less frequently, such as once a day, twice a day, thrice a day, once a week, twice a week, thrice a week, once every two weeks, twice every two weeks, thrice every two weeks, once a month, twice a month, thrice a month, or even less frequently, such as once every several months or even once or a few times a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the subject, etc. The formulations of the pharmaceutical compositions may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[0259] Individuals to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.

[0260] These compositions described herein are by no means all inclusive, and further modifications to suit the specific application will be apparent to the ordinary skilled artisan. Moreover, the effective amount of the compositions can be further approximated through analogy to compounds known to exert the desired effect. Prebiotics

[0261] In an aspect of the current disclosure, prebiotics are provided. In some embodiments, the prebiotics comprise one or more of the disclosed modified BSH enzymes. The prebiotics may further comprise one or more additional ingredients to stabilize or preserve the modified BSH enzymes of the instant disclosure.

[0262] As used herein, “prebiotic” refers to a nondigestible food ingredient that promotes the growth of beneficial microorganisms in the digestive tract of an animal (for example, in the intestines).

[0263] The prebiotics are believed to modif ' the gut microbiome of a subject by increasing the amount of unconjugated bile acids. Further, increasing the unconjugated bile acids may improve the uptake of lipids from foods in livestock thereby improving the growth and health of livestock.

[0264] As discussed above, the modified BSH enzymes of the instant disclosure have distinct properties and may be selected or combined based on those properties, e.g., activity, selectivity, activity in a particular pH, to be included in a prebiotic composition. The prebiotics may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different modified enzyme types, i.e., modified BSH enzymes comprising distinct substitutions.

[0265] Probiotics

[0266] In an aspect of the current disclosure, probiotics are provided. In some embodiments, the probiotics comprise the engineered bacterial cells of the instant disclosure. The probiotics may further comprise one or more carrier or excipient.

[0267] As used herein, “probiotic”’ refers to live microorganisms that are intended to have health benefits when consumed or applied to the body. Probiotics may be used in nutraceutical compositions, which are also disclosed herein. Probiotics may also be referred to as live biotherapeutic products (LBPs).

[0268] The formulation of probiotics, e.g., to be ingested or delivered, is known in the art and is considered routine.

[0269] As discussed above, the provision of the disclosed modified enzymes, e.g., in the form of a probiotic comprising an engineered bacterial cell expressing the modified enzyme, is believed to be effective at increasing the amount of unconjugated bile acids in the gut of subject, e.g. human subjects or non-human animal subjects.

[0270] As the disclosed modified enzymes have different properties, e.g., activities and, potentially, specificity, potentially at different pHs, the probiotics may comprise more than one type of engineered bacterial cell. For example, the probiotics may comprise more than one species of bacterial cell. The probiotics may comprise more a first type of engineered bacterial cell comprising a particular modified BSH enzyme and a second type of engineered bacterial cell comprising a second different modified BSH enzyme. The different types of engineered bacterial cells may be selected to occupy different niches in the microbiome of the host (subject).

[0271] The disclosed probiotics may be used in methods, e.g., the disclosed probiotics may be provided or administered to a subject.

[0272] The disclosed probiotics may be used in methods of improving the yield of a livestock animal, the methods comprising providing an effective amount of a probiotic of the instant disclosure to improve the yield of the livestock.

[0273] The livestock animal may be, e.g., a ruminant, a pig, a bird, a fish, a crustacean, or a mollusk. The ruminant may be, e.g., a cow, a sheep, a goat, a deer, a buffalo, or a camelid.

[0274] Medical foods

[0275] In some embodiments, the invention relates to medical foods. In some embodiments, the medical foods comprise the described BSH enzymes, the prebiotics, bacterial cells, or probiotics of the instant disclosure. In some embodiments, the invention relates to nutraceuticals, supplements, and GRAS designated products comprising the described BSH enzymes the prebiotics, bacterial cells, or probiotics of the instant disclosure.

[0276] As used herein, “"medical food” refers to a food which is formulated to be consumed or administered enterally under the supervision of a physician and which is intended for the specific dietary management of a disease or condition for which distinctive nutritional requirements, based on recognized scientific principles, are established by medical evaluation. The medical foods may be administered in any form including, but not limited to, capsules, pills, yogurt, drinks, powders, tablets, suspensions, aqueous solutions, emulsions, syrups, creams, pastes, gels, suppositories, ointments, sprays, foams, mills, colloids, etc.

[0277] Methods

[0278] The invention relates, in part, to methods comprising administering a pharmaceutical composition comprising a modified bile salt metabolism enzyme. In some embodiments, the modified bile salt metabolism enzyme has enhanced enzymatic activity, lowered enzymatic activity, enhanced substrate specificity and / or enhanced stability.

[0279] In some embodiments, the method comprises administering a modified bile salt metabolism enzyme to a subject in need thereof, wherein the modified bile salt metabolism enzyme is BSH, wherein the BSH comprises has at least 85% homology to an amino acid sequence set forth as SEQ ID NO: 31 to SEQ ID NO: 34. In some embodiments, the modified bile salt hydrolase (BSH) has at least 80% homology to an amino acid sequence set forth as SEQ ID NO: 31 to SEQ ID NO: 34. According to some embodiments, the above modified bile salt hydrolase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to an amino acid sequence set forth as SEQ ID NO: 31 to SEQ ID NO: 34.

[0280] In some embodiments, the method comprises administering a modified bile salt metabolism enzyme to a subject in need thereof, wherein the modified bile salt metabolism enzyme is BSH, wherein the BSH comprises at least one amino acid substitution at position L67, K195, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NOs: 31 or 33. In some embodiments, the least one substitution comprises L67Y, K195P, R297S, or Q328P, relative to SEQ ID NOs: 3 lor 33. In some embodiments, the method comprises administering a modified bile salt metabolism enzyme to a subject in need thereof, wherein the modified bile salt metabolism enzyme is BSH, wherein the BSH comprises at least one amino acid substitution at position Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 32. In some embodiments, the least one substitution comprises Q328P, relative to SEQ ID NO:32. In some embodiments, the method comprises administering a modified bile salt metabolism enzyme to a subject in need thereof, wherein the modified bile salt metabolism enzyme is BSH, wherein the BSH comprises at least one amino acid substitution at position Y34, H47, 157, L127, KI 89, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34. In some embodiments, the at least one substitution comprises Y34K, H47N, I57V, L127I, K189P, N292S, or R323P, relative to SEQ ID NO: 34. In some embodiments, the method further comprises administering bile acid acyl synthetase for succinyl (BAS-suc). In some embodiments, the bile acid acyl synthetase for succinyl (BAS-suc) comprises an amino acid sequence set forth as SEQ ID NO: 35. In some embodiments, the bile acid acyl synthetase for succinyl (BAS-suc) comprises an amino acid sequence set forth as SEQ ID NO: 50. In some embodiments, the method comprises administering BAS-Suc having an amino acid sequence set forth as SEQ ID NO: 50 and a BSH having an amino acid sequence set forth as SEQ ID NO: 36.

[0281] In some embodiments, the present invention provides a the method comprises administering a composition comprising a nucleic acid molecule encoding the modified BSH according to one or more of the above embodiments. In some embodiments, the composition comprises a nucleic acid molecule comprising a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence set forth as SEQ ID NOs: 41-44. In some embodiments, the composition comprises a nucleic acid molecule comprising a nucleic acid sequence encoding a modified bile salt hydrolase (BSH) enzyme at least 90% identical to SEQ ID NOs: 31 or 33 and comprising one or more amino acid substitution at position L67, K195, R297, or Q328, relative to SEQ ID NOs: 31 or 33. In some embodiments, the one or more substitutions comprise L67Y, K195P, R297S, or Q328P, relative to SEQ ID NOs: 31 or 33. In some embodiments, the composition comprises a nucleic acid molecule comprising a nucleic acid sequence encoding a modified bile salt hydrolase (BSH) enzyme at least 90% identical to SEQ ID NO: 32 and comprising at least one amino acid substitution at position Q328, relative to SEQ ID NO:32. In some embodiments, the at least one substitution comprises Q328P, relative to SEQ ID NO: 32. In some embodiments, the composition comprises a nucleic acid molecule comprising a nucleic acid sequence encoding a modified bile salt hydrolase (BSH) enzyme at least 90% identical to SEQ ID NO: 34 and comprising one or more amino acid substitution at position Y34, H47, 157, L127, K189, N292, or R323, relative to SEQ ID NO: 34. In some embodiments, the one or more substitutions comprise Y34K, H47N, I57V, L127I, K189P, N292S, or R323P, relative to SEQ ID NO: 34. In some embodiments, the composition comprises a nucleic acid molecule comprising a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence set forth as SEQ ID NOs: 16-30 or 51-55. In some embodiments, the composition comprises a nucleic acid molecule comprising a nucleic acid sequence encoding bile acid acyl synthetase for succinyl (BAS-suc). In one embodiment, the composition comprises a nucleic acid molecule comprising a nucleic acid sequence encoding bile acid acyl synthetase for succinyl (BAS-suc) at least 80% identical to SEQ ID NO: 35 or SEQ ID NO: 50. In some embodiments, the composition comprises a nucleic acid molecule comprising a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 45 or SEQ ID NO: 46.

[0282] In another aspect, the invention relates to methods comprising administering a pharmaceutical composition comprising a genetically modified bacterial cell and at least one carrier, wherein the modified bacterial cell expresses one or more heterologous genes encoding at least one bile salt metabolism enzyme.

[0283] In some embodiments, the method comprises administering a genetically modified bacterial cell expressing one or more heterologous genes encoding at least one bile salt metabolism enzyme to a subject in need thereof, wherein the at least one bile salt metabolism enzyme is BSH, wherein the BSH comprises at least one amino acid substitution at position L67, K195, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NOs: 31 or 33. In some embodiments, the least one substitution comprises L67Y, K195P, R297S, or Q328P, relative to SEQ ID NOs: 31 or 33. In some embodiments, the method comprises administering a genetically modified bacterial cell expressing one or more heterologous genes encoding at least one bile salt metabolism enzyme to a subject in need thereof, wherein the at least one bile salt metabolism enzyme is BSH, wherein the BSH comprises at least one amino acid substitution at position Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 32. In some embodiments, the least one substitution comprises Q328P, relative to SEQ ID NO: 32. In some embodiments, the method comprises administering a genetically modified bacterial cell expressing one or more heterologous genes encoding at least one bile salt metabolism enzyme to a subject in need thereof, wherein the at least one bile salt metabolism enzyme is BSH, wherein the BSH comprises at least one amino acid substitution at position Y34, H47, 157, L127, KI 89, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34. In some embodiments, the at least one substitution comprises Y34K, H47N, I57V, L127I, K189P, N292S, or R323P, relative to SEQ ID NO: 34. In some embodiments, the method further comprises administering a genetically modified bacterial cell expressing one or more heterologous genes encoding bile acid acyl synthetase for succinyl (BAS-suc). In some embodiments, the one or more heterologous genes encoding bile acid acyl synthetase for succinyl (BAS-suc) encode an amino acid sequence comprising SEQ ID NO: 35. In some embodiments, the one or more heterologous genes encoding bile acid acyl synthetase for succinyl (BAS-suc) encode an amino acid sequence comprising SEQ ID NO: 50. In some embodiments, the genetically modified bacterial cell expresses one or more heterologous genes encoding a BAS-Suc having an amino acid sequence set forth as SEQ ID NO: 50 and a BSH having an amino acid sequence set forth as SEQ ID NO: 36.

[0284] In some embodiments, the genetically modified bacterial cell is Bacteroides thetaiotaomicron. In some embodiments, the Bacteroides thetaiotaomicron comprises a knockout mutation in at least one gene encoding a bile acid or bile salt metabolism enzyme. In some embodiments, the at least one gene encoding a bile acid or bile salt metabolism enzyme is selected from the group consisting of: bshA, bshB, hsdhA, or any combination thereof.

[0285] In some embodiments, the method comprises administrating a pharmaceutical composition comprising a modified bile salt metabolism enzyme or a genetically modified bacterial cell and at least one carrier, wherein the modified bacterial cell expresses one or more heterologous genes encoding at least one bile salt metabolism enzyme. In some embodiments, the pharmaceutical composition comprises the modified bacterial cell expressing one or more heterologous genes encoding at least one bile salt metabolism enzyme at any concentration. In some embodiments, the pharmaceutical composition comprises the modified bacterial cell expressing one or more heterologous genes encoding at least one bile salt metabolism enzyme at a concentration of about 1 x 103colony forming units (CFUs) or below. In some embodiments, the modified bacterial cell expressing one or more mutated heterologous genes encoding at least one bile salt metabolism enzyme is administered at a concentration of about 1 xlO13CFU / animal or higher. In some embodiments, the pharmaceutical composition comprises the modified bacterial cell expressing one or more heterologous genes encoding at least one bile salt metabolism enzyme at a concentration of about at least 1 x 103colony forming units (CFUs), 1 x 104colony forming units (CFUs), 1 x 105colony forming units (CFUs), 5 x 105colony forming units (CFUs), 1 x 106colony forming units (CFUs), 2 x 106colony forming units (CFUs), 3 x 106colony forming units (CFUs), 4 x 106colony forming units (CFUs), 5 x 106colony forming units (CFUs), 6 x 106colony forming units (CFUs), 7 x 106colony forming units (CFUs), 8 x 106colony forming units (CFUs), 9 x

[0286] 106colony forming units (CFUs), 1 x 107colony forming units (CFUs), 2 x 107colony forming units (CFUs), 3 x 107colony forming units (CFUs), 4 x 107colony forming units (CFUs), 5 x 107colony forming units (CFUs), 6 x 107colony forming units (CFUs), 7 x

[0287] 107colony forming units (CFUs), 8 x 107colony forming units (CFUs), 9 x 107colony forming units (CFUs), 1 x 108colony forming units (CFUs), 2 x 108colony forming units (CFUs), 3 x 108colony forming units (CFUs), 4 x 108colony forming units (CFUs), 5 x

[0288] 108colony forming units (CFUs), 6 x 108colony forming units (CFUs), 7 x 108colony forming units (CFUs), 8 x 108colony forming units (CFUs), 9 x 108colony forming units (CFUs), 1 x 109colony forming units (CFUs), 5 x 109colony forming units (CFUs), 1 x IO10colony forming units (CFUs) 5 x IO10colony forming units (CFUs), 1 x 1011colony forming units (CFUs) 5 x 1011colony forming units (CFUs), 1 x 1012colony forming units (CFUs) 5 x 1012colony forming units (CFUs), or 1 x 1013colony forming units (CFUs). In some embodiments, the modified bacterial cell expressing one or more mutated heterologous genes encoding at least one bile salt metabolism enzyme is administered at a concentration of about 1 xlO13CFU / animal. In some embodiments, the modified bacterial cell expressing one or more mutated heterologous genes encoding at least one bile salt metabolism enzyme is administered at a concentration of about IxlO9CFU / animal.

[0289] In some embodiments, the pharmaceutical composition comprises the modified bacterial cell expressing one or more heterologous genes encoding at least one bile salt metabolism enzyme at a concentration of about at least 5% by weight of the composition. In some embodiments, the pharmaceutical composition comprises the modified bacterial cell at a concentration ranging from about 0.1% to 99.9% by weight of the composition.

[0290] In some embodiments, the pharmaceutical composition is administered, orally, rectally, intravenously, intranasally, ocularly, subcutaneously, transdermally, or transmucosally. In some embodiments, the modified bacterial cell expressing one or more mutated heterologous genes encoding at least one bile salt metabolism enzyme is administered about twice a week.

[0291] In some embodiments, the pharmaceutical composition is administered according to a dosing regimen selected from: once daily (QD), twice daily (BID), three times daily (TID), four times daily (QID), once every other day (QOD), twice a week, three times a week, four times a week, five times a week, six times a week, once weekly (weekly), once every 10 days, once every two weeks (biweekly), twice monthly, once monthly, once every six weeks, once every two months, once every three months (quarterly), twice yearly, or once yearly. In certain embodiments, the composition may be administered as a single dose or as multiple divided doses within a 24-hour period. The specific dosing frequency may be adjusted based on factors such as the pharmacokinetics of the compound, the route of administration, patient compliance, disease severity, or therapeutic goals.

[0292] Methods of Increasing Bile Acid Deconjugation

[0293] The invention relates, in part, to methods of increasing bile acid deconjugation. Bile acid deconjugation is the process by which conjugated bile acids (which are bile acids bound to amino acids like glycine or taurine) are converted back into their unconjugated form by the action of microbial enzymes in the gut, particularly bile salt hydrolases (BSHs) produced by intestinal bacteria. The bile salt metabolism enzymes described herein are characterized by having an enhanced enzymatic activity, lowered enzymatic activity, enhanced substrate specificity and / or enhanced stability.

[0294] In some embodiments, the method of increasing bile acid deconjugation comprises administering a composition comprising a modified bile salt metabolism enzyme. In some embodiments, the method of increasing bile acid deconjugation comprises administering a modified bile salt metabolism enzyme to a subject in need thereof, wherein the modified bile salt metabolism enzyme is BSH, wherein the BSH comprises at least one amino acid substitution at position L67, K195, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NOs: 31 or 33. In some embodiments, the least one substitution comprises L67Y, K195P, R297S, or Q328P, relative to SEQ ID NOs: 31 or 33. In some embodiments, the method of increasing bile acid deconjugation comprises administering a modified bile salt metabolism enzyme to a subject in need thereof, wherein the modified bile salt metabolism enzyme is BSH, wherein the BSH comprises at least one amino acid substitution at position Q328, when compared to the amino acid sequence set forth as SEQ ID NO:32. In some embodiments, the least one substitution comprises Q328P, relative to SEQ ID NO:32. In some embodiments, the method of increasing bile acid deconjugation comprises administering a modified bile salt metabolism enzyme to a subject in need thereof, wherein the modified bile salt metabolism enzyme is BSH, wherein the BSH comprises at least one amino acid substitution at position Y34, H47, 157, L127, K189, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34. In some embodiments, the at least one substitution comprises Y34K, H47N, I57V, LI 271, K189P, N292S, or R323P, relative to SEQ ID NO: 34. In some embodiments, the method of increasing bile acid deconjugation further comprises administering bile acid acyl synthetase for succinyl (BAS-suc). In some embodiments, the bile acid acyl synthetase for succinyl (BAS-suc) comprises an amino acid sequence comprising SEQ ID NO: 35. In some embodiments, the bile acid acyl synthetase for succinyl (BAS-suc) comprises an amino acid sequence comprising SEQ ID NO: 50. In some embodiments, the method comprises administering a BAS-Suc having an amino acid sequence set forth as SEQ ID NO: 50 and a BSH having an amino acid sequence set forth as SEQ ID NO: 36. In some embodiments, the method of increasing bile acid deconjugation comprises administering a composition comprising a genetically modified bacterial cell and at least one carrier, wherein the modified bacterial cell expresses one or more heterologous genes encoding at least one bile salt metabolism enzyme. In some embodiments, the method of increasing bile acid deconjugation comprises administering a genetically modified bacterial cell expressing one or more heterologous genes encoding at least one bile salt metabolism enzyme, wherein the at least one bile salt metabolism enzyme is BSH. In some embodiments, the one or more heterologous genes encoding native bile salt hydrolase (BSH) encode an amino acid sequence comprising SEQ ID NO: 34, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33.

[0295] In some embodiments, the method of increasing bile acid deconjugation comprises administering a genetically modified bacterial cell expressing one or more heterologous genes encoding at least one bile salt metabolism enzyme, wherein the at least one bile salt metabolism enzyme is BSH, wherein the BSH comprises at least one amino acid substitution at position L67, KI 95, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NOs: 31 or 33. In some embodiments, the least one substitution comprises L67Y, K195P, R297S, or Q328P, relative to SEQ ID NOs: 31 or 33. In some embodiments, the method of increasing bile acid deconjugation comprises administering a genetically modified bacterial cell expressing one or more heterologous genes encoding at least one bile salt metabolism enzyme, wherein the at least one bile salt metabolism enzyme is BSH, wherein the BSH comprises at least one amino acid substitution at position Q328, when compared to the amino acid sequence set forth as SEQ ID NO:32. In some embodiments, the least one substitution comprises Q328P, relative to SEQ ID NO: 32. In some embodiments, the method of increasing bile acid deconjugation comprises administering a genetically modified bacterial cell expressing one or more mutated heterologous genes encoding at least one bile salt metabolism enzyme to a subject in need thereof, wherein the at least one bile salt metabolism enzyme is BSH, wherein the BSH comprises at least one amino acid substitution at position Y34, H47, 157, L127, KI 89, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34. In some embodiments, the at least one substitution comprises Y34K, H47N, I57V, L127I, K189P, N292S, or R323P, relative to SEQ ID NO: 34. In some embodiments, the method of increasing bile acid deconjugation further comprises administering a genetically modified bacterial cell expressing one or more heterologous genes encoding bile acid acyl synthetase for succinyl (BAS-suc). In some embodiments, the one or more heterologous genes encoding bile acid acyl synthetase for succinyl (BAS-suc) encode an amino acid sequence comprising SEQ ID NO: 35. In some embodiments, the one or more heterologous genes encoding bile acid acyl synthetase for succinyl (BAS-suc) encode an amino acid sequence comprising SEQ ID NO: 50. In some embodiments, the genetically modified bacterial cell expresses one or more heterologous genes encoding a BAS-Suc having an amino acid sequence set forth as SEQ ID NO: 50 and a BSH having an amino acid sequence set forth as SEQ ID NO: 36. In some embodiments, the genetically modified bacterial cell is Bacteroides thetaiotaomicron. In some embodiments, the Bacteroides thetaiotaomicron comprises a knockout mutation in at least one gene encoding a bile acid or bile salt metabolism enzyme. In some embodiments, the at least one gene encoding a bile acid or bile salt metabolism enzyme is selected from the group consisting of: bshA, bshB, hsdhA, or any combination thereof.

[0296] In some embodiments, the bile acid to be deconjugated is a primary or secondary bile acid. Non-limiting examples of primary bile acids include, glycocholic acid (GCA), taurocholic acid (TCA), glycochenodeoxycholic acid (GCDCA), and taurochenodeoxycholic acid (TCDCA). Non-limiting examples of secondary bile acids include, glycodeoxy cholic acid (GDCA), taurodeoxy cholic acid (TDCA), glycolithocholic acid (GLCA), and taurolithocholic acid (TLCA).

[0297] In some embodiments, the method of increasing bile acid deconjugation comprises administering at least one modified BSH to target a specific pool of BAs. In some embodiments, the method of increasing bile acid deconjugation of GCA, TCA, GCDCA, TCDCA, GDCA, TDCA, GLCA, or TLCA comprises administering a composition comprising a modified BSH, wherein the modified BSH comprises an amino acid as set forth as SEQ ID NO:7 and SEQ ID NOTO. In some embodiments, the method of increasing bile acid deconjugation of non-deoxycholate BAs comprises administering a composition comprising a modified BSH, wherein the modified BSH comprises an amino acid as set forth as SEQ ID NO:4. In some embodiments, the method of increasing bile acid deconjugation of deoxycholate BAs comprises administering a composition comprising a modified BSH, wherein the modified BSH comprises an amino acid as set forth as SEQ ID NO: 13.

[0298] Methods of Treatment

[0299] The invention relates, in part, to methods of treating a condition associated with impaired metabolism. In some embodiments, the condition is associated with impaired lipid metabolism. In some embodiments, the method comprises administering to a subject in need of such treatment an effective amount of the composition according to one or more of the above embodiments.

[0300] In some embodiments, the method of treating a condition associated with impaired metabolism comprises administering to a subject at least one modified BSH to target a diverse pool of BAs. In some embodiments, the method comprises administering at least one modified BSH to target a specific pool of BAs. In some embodiments, the method comprises administering at least one modified BSH to target a single species of BA. In some embodiments, the subject is administered a tailored BSH composition comprising one or more modified BSH to treat a condition associated with impaired metabolism.

[0301] In some embodiments, the method of treating a condition associated with impaired metabolism comprises administering a pharmaceutical composition comprising a modified bile salt metabolism enzyme. In some embodiments, the method of treating a condition associated with impaired metabolism comprises administering a modified bile salt metabolism enzyme to a subject in need thereof, wherein the modified bile salt metabolism enzyme is BSH, wherein the BSH comprises at least one amino acid substitution at position L67, K195, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NOs: 31 or 33. In some embodiments, the least one substitution comprises L67Y, K195P, R297S, or Q328P, relative to SEQ ID NOs: 31 or 33. In some embodiments, the method of treating a condition associated with impaired metabolism comprises administering a modified bile salt metabolism enzyme to a subject in need thereof, wherein the modified bile salt metabolism enzyme is BSH, wherein the BSH comprises at least one amino acid substitution at position Q328, when compared to the amino acid sequence set forth as SEQ ID NO:32. In some embodiments, the least one substitution comprises Q328P, relative to SEQ ID NO:32. In some embodiments, the method of treating a condition associated with impaired metabolism comprises administering a modified bile salt metabolism enzyme to a subject in need thereof, wherein the modified bile salt metabolism enzyme is BSH, wherein the BSH comprises at least one amino acid substitution at position Y34, H47, 157, L127, K189, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34. In some embodiments, the at least one substitution comprises Y34K, H47N, I57V, L127I, K189P, N292S, or R323P, relative to SEQ ID NO: 34. In some embodiments, the method further comprises administering a bile acid acyl synthetase for succinyl (BAS-suc). In some embodiments, the bile acid acyl synthetase for succinyl (BAS-suc) comprises an amino acid sequence comprising SEQ ID NO: 35. In some embodiments, the bile acid acyl synthetase for succinyl (BAS-suc) comprises an amino acid sequence comprising SEQ ID NO: 50. In some embodiments, the method comprises administering a BAS-Suc having an amino acid sequence set forth as SEQ ID NO: 50 and a BSH having an amino acid sequence set forth as SEQ ID NO: 36.

[0302] In some embodiments, the method of treating a condition associated with impaired metabolism comprises administering a pharmaceutical composition comprising a genetically modified bacterial cell and at least one carrier, wherein the modified bacterial cell expresses one or more heterologous genes encoding at least one bile salt metabolism enzyme.

[0303] In some embodiments, the method of treating a condition associated with impaired metabolism comprises administering a composition comprising a genetically modified bacterial cell and at least one carrier, wherein the modified bacterial cell expresses one or more heterologous genes encoding at least one bile salt metabolism enzyme. In some embodiments, the method of treating a condition associated with impaired metabolism comprises administering a genetically modified bacterial cell expressing one or more heterologous genes encoding at least one bile salt metabolism enzyme, wherein the at least one bile salt metabolism enzyme is BSH. In some embodiments, the one or more heterologous genes encoding native bile salt hydrolase (BSH) encode an amino acid sequence comprising SEQ ID NO: 34, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33. In some embodiments, the method of treating a condition associated with impaired metabolism comprises administering a genetically modified bacterial cell expressing one or more heterologous genes encoding at least one bile salt metabolism enzyme to a subject in need thereof, wherein the at least one bile salt metabolism enzyme is BSH, wherein the BSH comprises at least one amino acid substitution at position L67, KI 95, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NOs: 31 or 33. In some embodiments, the least one substitution comprises L67Y, K195P, R297S, or Q328P, relative to SEQ ID NOs: 31 or 33. In some embodiments, the method of treating a condition associated with impaired metabolism comprises administering a genetically modified bacterial cell expressing one or more heterologous genes encoding at least one bile salt metabolism enzyme to a subject in need thereof, wherein the at least one bile salt metabolism enzyme is BSH, wherein the BSH comprises at least one amino acid substitution at position Q328, when compared to the amino acid sequence set forth as SEQ ID NO:32. In some embodiments, the least one substitution comprises Q328P, relative to SEQ ID NO :32. In some embodiments, the method of treating a condition associated with impaired metabolism comprises administering a genetically modified bacterial cell expressing one or more heterologous genes encoding at least one bile salt metabolism enzyme to a subject in need thereof, wherein the at least one bile salt metabolism enzyme is BSH, wherein the BSH comprises at least one amino acid substitution at position Y34, H47, 157, L127, K189, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34. In some embodiments, the at least one substitution comprises Y34K, H47N, I57V, L127I, K189P, N292S, or R323P, relative to SEQ ID NO: 34. In some embodiments, the method further comprises administering a genetically modified bacterial cell expressing one or more heterologous genes encoding bile acid acyl synthetase for succinyl (BAS-suc). In some embodiments, the one or more heterologous genes encoding bile acid acyl synthetase for succinyl (BAS-suc) encode an amino acid sequence comprising SEQ ID NO: 35. In some embodiments, the one or more heterologous genes encoding bile acid acyl synthetase for succinyl (BAS-suc) encode an amino acid sequence comprising SEQ ID NO: 50. In some embodiments, the genetically modified bacterial cell expresses one or more heterologous genes encoding a BAS-Suc having an amino acid sequence set forth as SEQ ID NO: 50 and a BSH having an amino acid sequence set forth as SEQ ID NO: 36.

[0304] In some embodiments, the genetically modified bacterial cell is Bacteroides thetaiotaomicron . In some embodiments, the Bacteroides thetaiotaomicron comprises a knockout mutation in at least one gene encoding a bile acid or bile salt metabolism enzyme. In some embodiments, the at least one gene encoding a bile acid or bile salt metabolism enzyme is selected from the group consisting of: bshA, bshB, hsdhA, or any combination thereof.

[0305] In some embodiments, the subject is a mammalian subject. In some embodiments, the subject in need thereof or subject may refer to a vertebrate animal, e.g., livestock, e.g., cow. chicken, goat, sheep, llama, alpaca, camel, horse, donkey, fish, crustacean, mollusk, etc., a human, a companion animal, e.g., dog. cat, hamster, rat. chinchilla, ferret, etc., a laboratory animal, e.g., mouse, rat, rabbit, monkey, ape, etc. In some embodiments, the subject is a human. In some embodiments, the subject is a cat.

[0306] In some embodiments, the subject has a condition associated with impaired metabolism. A non-limiting list of conditions associated with impaired metabolism includes metabolic-associated steatohepatitis (MASH), cholestatic disease, liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), metabolic disorder, metabolic syndrome, cardiovascular disease, hyperlipidemia, dyslipidemia, insulin resistance, type II diabetes, impaired glucose tolerance, obesity, cholestatic disease, or any combination thereof. In some embodiments, the condition associated with impaired metabolism is selected from a group consisting of: metabolic-associated steatohepatitis (MASH), cholestatic disease, liver disease, nonalcoholic fatty liver disease (NAFLD / MASLD), and non-alcoholic steatohepatitis (NASH). In some embodiments, the condition associated with impaired metabolism is cardiovascular disease.

[0307] In some embodiments, the composition is administered in combination with at least one therapeutically active agent. As used herein, the phrase “in combination with at least one therapeutically active agent’” refers, without limitation, to a “therapeutic combination” which is meant to be understood, as a combination of a number of components that is administered to a subject in need to provide a desirable therapeutic effect. In the combination according to the embodiments of the invention, the at least two components can be given in a single formulation or each as a separate medicament; they can be administered simultaneously, or alternatively, can have each a specific dosing regimen and / or administration regime. The combination according to the embodiments of the invention can be a synergistic combination, while the combined effect is larger than the additive effect of each individual drug. In one embodiment, the combination has an additive effect on the clinical outcome.

[0308] In some embodiments, the invention provides a method of alleviating symptoms of a disease or condition associated with impaired metabolism. According to some embodiments, the above method comprising administering to a subject afflicted with said disease or condition an effective amount of the composition according to one or more of the above embodiments. In the context of the invention, the term “symptom” refers, without limitation, to signs and clinical manifestations of the disease that can be either objective, when observed by a physician, or subjective, when perceived by the patient. The term “alleviating a symptom” refers, without limitation, to elimination and / or reduction and / or decreased performance of the symptoms of the disease or condition.

[0309] In some embodiments, the invention provides a method of preventing and / or delaying an onset and / or or preventing and / or slowing progression of a disease or condition associated with impaired metabolism. According to some embodiments, the above method comprising administering to a subject afflicted with said disease or condition an effective amount of the composition according to one or more of the above embodiments. As used herein, the phrase “slowing and / or preventing progression” refers, without limitation, to the influence of the treatment on the clinical course of the disease. The “slowing and / or preventing” progression of the condition according to the embodiments of the above method may be measured using any appropriate questionary, method, scale, diagnostic tool, or any other means that are known in the art or acceptable by the relevant functions and professionals. The term “preventing” might but does not necessarily mean recovery from the illness. As such, the term “preventing” relates to the situation when the patient does not present symptoms and / or signs and / or manifestations of the next “stage” of illness severity as defined by the appropriate and acceptable parameters for the specific disease condition. The term “slowing”, or atenuating is can, without limitation, prolong the time of transition into the next “stage” of illness severity, thus providing greater window of opportunity for extensive care and recovery. According to some embodiments, the above method comprises administering to a subject in need of such treatment an effective amount of the composition according to one or more of the above embodiments. A non-limiting list of conditions associated with impaired metabolism includes metabolic disorder, metabolic syndrome, cardiovascular disease, cholestatic disease, hyperlipidemia, dyslipidemia, insulin resistance, type II diabetes, impaired glucose tolerance, obesity, liver disease, NAFLD / NASH, MASLD / MASH, cholestatic disease, or any combination thereof.

[0310] Methods of Manufacture

[0311] The invention relates, in part, to methods of manufacturing a composition comprising the compositions described herein. In some embodiments, the methods of manufacutring a modified bile salt hydrolase (BSH) enzyme are provided and comprise introducing a polynucleotide of the instant disclosure into a cell, thereby causing the cell to express the modified BSH enzyme. In some embodiments, the methods further comprise enriching, purifying, or isolating the modified BSH enzyme. In some embodiments, the cell is a mammalian cell, an insect cell, a fungal cell, or a bacterial cell. In some embodiments, the method of manufacture comprises: a) generating genetically modified Bacteroides thetaiotaomicron strain; b) transforming the genetically modified Bacteroides thetaiotaomicron with at least one vector containing one or more heterologous genes encoding at least one bile salt metabolism enzyme; c) expressing the at least one bile salt metabolism enzyme in the genetically modified Bacteroides thetaiotaomicron^ and, d) preparing the composition comprising the genetically modified Bacteroides thetaiotaomicron and at least one carrier. Abacterial strain may be modified by any method known by one skilled in the art. Bacteria may be transformed by any method known by one skilled in the art.

[0312] EXEMPLARY EMBODIMENTS

[0313] This invention provides the following non-limiting embodiments. Embodiment l is a modified bile salt hydrolase (BSH) having at least 80% homology to an amino acid sequence set forth as SEQ ID NO: 34, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33 and comprising one or more substitutions relative to SEQ ID NO: 34, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33.

[0314] Embodiment 2 is the modified BSH of embodiments 1, wherein the modified BSH comprises at least 90% homology to an amino acid sequence set forth as SEQ ID NO: 34, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33 and comprising one or more substitutions relative to SEQ ID NO: 34, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33.

[0315] Embodiment 3 is the modified BSH of embodiment 1 or 2, wherein the modified BSH comprises an amino acid sequence set forth as SEQ ID NO: 7, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 4, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0316] Embodiment 4 is the modified BSH of any one of embodiments 1 through

[0317] 3, wherein the modified BSH comprises at least one amino acid substitution at position L67, K195, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 31 or 33.

[0318] Embodiment 5 is the modified BSH of any one of embodiments 1 through

[0319] 4, wherein the modified BSH comprises a combination of at least two amino acid substitutions at any of the positions L67, K195, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 31 or 33.

[0320] Embodiment 6 is the modified BSH of any one of embodiments 1 through

[0321] 5, wherein the modified BSH comprises a combination of multiple amino acid substitutions at any of the positions L67, K195, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 31 or 33.

[0322] Embodiment 7 is the modified BSH of any one of embodiments 1 through

[0323] 6, wherein the modified BSH comprises at least one amino acid substitution at position Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 32.

[0324] Embodiment 8 is the modified BSH of any one of embodiments 1 through

[0325] 7, wherein the modified BSH comprises at least one amino acid substitution at position Y34, H47, 157, LI 27, KI 89, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34.

[0326] Embodiment 9 is the modified BSH of any one of embodiments 1 through 8, wherein the modified BSH comprises at least two amino acid substitutions at any of the positions Y34, H47, 157, L127, K189, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34.

[0327] Embodiment 10 is the modified BSH of any one of embodiments 1 through 9, wherein the modified BSH comprises a combination of multiple amino acid substitutions at any of the positions Y34, H47, 157, L127, KI 89, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34.

[0328] Embodiment 11 is the modified BSH of any one of embodiments 1 through 10, wherein the modified BSH is expressed in a modified bacterial cell.

[0329] Embodiment 12 is the modified BSH of any one of embodiments 1 through 11, wherein the modified bacterial cell is Bacteroides thetaiotaomicron.

[0330] Embodiment 13 is an isolated oligonucleotide having at least 80% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 41 to 44.

[0331] Embodiment 14 is the isolated oligonucleotide of embodiments 13, wherein the isolated oligonucleotide comprises at least 90% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 41 to 44.

[0332] Embodiments 15 is the isolated oligonucleotide of embodiments 13 or 14, wherein the isolated oligonucleotide comprises a polynucleotide sequence set forth as SEQ ID NO: 16 to 30 or SEQ ID NO: 51 to 55.

[0333] Embodiment 16 is an expression vector comprising the isolated oligonucleotide of any of any one of embodiments 13-15 operably linked to at least one promotor.

[0334] Embodiment 17 is a composition comprising an isolated oligonucleotide of any one of embodiments 13-15, an expression vector of embodiment 16, a modified BSH of any one of embodiments 1-12, or any combination thereof, and at least one carrier. Embodiment 18 is a composition of embodiment 17, further comprising a bile acid acyl synthetase for succinyl (BAS-suc) comprising an amino acid sequence set forth as SEQ ID NO: 35, SEQ ID NO: 50, or a homologue thereof.

[0335] Embodiment 19 is the composition of embodiment 17 or 18 for use as a medicament.

[0336] Embodiment 20 is the composition of embodiment 17 or 18, wherein the composition is a nutraceutical, supplement, medical food, and / or GRAS designated products.

[0337] Embodiment 21 is the composition of embodiment 17 or 18 for use in the treatment of a disease or condition associated with impaired metabolism.

[0338] Embodiment 22 is a method of treating a disease or condition associated with impaired metabolism, comprising administering to the subject in need an effective amount of the composition of embodiment 17 or 18.

[0339] Embodiment 23 is a method of treating a condition associated with impaired metabolism and / or lipid metabolism, comprising administering to a subject in need of such treatment an effective amount of the composition of embodiment 17 or 18.

[0340] Embodiment 24 is a method of alleviating at least one symptom of a disease or condition associated with impaired metabolism comprising administering to a subject afflicted with said disease or condition an effective amount of the composition of embodiment 17 or 18.

[0341] Embodiment 25 is the method of embodiment 24, wherein the disease or condition is associated with impaired lipid metabolism.

[0342] Embodiment 26 is a method of preventing and / or delaying an onset and / or or preventing and / or slowing progression of a disease or condition associated with impaired metabolism comprising administering to a subject an amount of the composition of embodiment 17 or 18 effective to prevent or delay progression of a disease or condition associated with impaired metabolism.

[0343] Embodiment 27 is the method of embodiment 26, wherein the disease or condition is associated with impaired lipid metabolism.

[0344] Embodiment 28 is the method of any one of embodiments 22 through 27, wherein the condition associated with impaired metabolism is selected from the group consisting of a metabolic disorder, metabolic syndrome, cardiovascular disease, cholestatic disease, hyperlipidemia, dyslipidemia, insulin resistance, type II diabetes, impaired glucose tolerance, obesity, liver disease, NAFLD / MASLD, NASH / MASH, cholestatic disease, or any combination thereof.

[0345] Embodiment 29 is the method of any one of embodiments 22 through 27, wherein the composition is administered orally, rectally, intravenously, intranasally, ocularly, subcutaneously, transdennally, or transmucosally.

[0346] Embodiment 30 is a method for increasing bile acid deconjugation, wherein the method comprises administering the composition of embodiment 17 or 18.

[0347] Embodiment 31 is the method of embodiment 30, wherein the method targets a specific species of BAs.

[0348] Embodiment 32 is the method of embodiment 30, wherein the method increases deconjugation of circulating bile acids.

[0349] Embodiment 33 is a pharmaceutical composition comprising a modified bile salt hydrolase (BSH) enzyme, formulated in a capsule or pill or any other delivery method, wherein the composition is effective to modulate bile acid metabolism in a subject.

[0350] Embodiment 34 is the pharmaceutical composition of embodiment 33, wherein the modified BSH is administered at a concentration of about 0.1% to 100% by weight of the composition.

[0351] Embodiment 35 is a composition comprising a genetically modified Bacteroides thetaiotaomicron and at least one carrier, wherein the genetically modified Bacteroides thetaiotaomicron expresses one or more heterologous genes encoding at least one bile salt metabolism enzyme; and wherein the bile salt metabolism enzyme encoded by the one or more heterologous genes is characterized by having enhanced enzymatic activity, lower or lack of activity, and / or substrate specificity and / or stability.

[0352] Embodiment 36 is the composition of embodiment 35, wherein the genetically modified Bacteroides thetaiotaomicron bears a knockout mutation in at least one of the genes encoding bile acid or bile salt metabolism enzyme.

[0353] Embodiment 37 is the composition of embodiment 35 or 36, wherein the at least one bile salt metabolism enzyme is a bile salt hydrolase (BSH). Embodiment 38 is the composition of any one of embodiments 35 through

[0354] 37, wherein the BSH is a native Christenseriella minuta BSH.

[0355] Embodiment 39 is the composition of any one of embodiments 35 through

[0356] 38, wherein the at least one bile salt metabolism enzyme comprises an amino acid sequence set forth as SEQ ID NO: 34, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33.

[0357] Embodiment 40 is the composition of any one of embodiments 35 through

[0358] 39, wherein the at least one bile salt metabolism enzyme comprises an amino acid sequence set forth as SEQ ID NO: 7, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 4, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0359] Embodiment 41 is the composition of any one of embodiments 35 through

[0360] 40, wherein the genetically modified Bacteroides thetaiotaomicron expresses a combination of two or more heterologous genes encoding two or more bile salt metabolism enzymes, and wherein the two or more bile salt metabolism enzymes are characterized by having enhanced enzymatic activity, lower or lack of activity, and / or substrate specificity and / or stability, and, wherein the two or more bile salt metabolism enzymes, independently of each other, are characterized by having an amino acid sequence set forth as SEQ ID NO: 34, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, EQ ID NO: 7, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 4, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0361] Embodiment 42 is the composition of any one of embodiments 35 through

[0362] 41, wherein the BSH comprises at least one amino acid substitution at position L67, KI 95, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 31 or 33.

[0363] Embodiment 43 is the composition of any one of embodiments 35 through

[0364] 42, wherein the BSH comprises a combination of at least two amino acid substitutions at any of the positions L67, K195, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 31 or 33. Embodiment 44 is the composition of any one of embodiments 35 through

[0365] 43, wherein the BSH comprises a combination of multiple amino acid substitutions at any of the positions L67, K195, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 31 or 33.

[0366] Embodiment 45 is the composition of any one of embodiments 35 through

[0367] 44, wherein the BSH comprises at least one amino acid substitution at position Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 32.

[0368] Embodiment 46 is the composition of any one of embodiments 35 through

[0369] 45, wherein the BSH comprises at least one amino acid substitution at position Y34, H47, 157, L127, KI 89, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34.

[0370] Embodiment 47 is the composition of any one of embodiments 35 through

[0371] 46, wherein the BSH comprises a combination of at least two amino acid substitutions at any of the positions Y34, H47, 157, L127, K189, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34.

[0372] Embodiment 48 is the composition of any one of embodiments 35 through

[0373] 47, wherein the BSH comprises a combination of multiple amino acid substitutions at any of the positions Y34, H47, 157, L127, KI 89, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34.

[0374] Embodiment 49 is the composition of any one of embodiments 35 through

[0375] 48, wherein the one or more heterologous genes encoding at least one bile salt metabolism enzyme comprise a polynucleotide sequence having at least 80% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 16 to SEQ ID NO:30 or SEQ ID NO: 51 to SEQ ID NO: 55.

[0376] Embodiment 50 is the composition of any one of embodiments 35 through

[0377] 49, wherein the one or more heterologous genes encoding at least one bile salt metabolism enzyme comprise a polynucleotide sequence having at least 90% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 16 to SEQ ID NO:30 or SEQ ID NO: 51 to SEQ ID NO: 55.

[0378] Embodiment 51 is the composition of any one of embodiments 35 through

[0379] 50, wherein the one or more heterologous genes encoding at least one bile salt metabolism enzyme comprise a polynucleotide sequence having between 80% to 99% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 16 to SEQ ID NO:30 or SEQ ID NO: 51 to SEQ ID NO: 55.

[0380] Embodiment 52 is the composition of any one of embodiments 35 through

[0381] 51, wherein the one or more heterologous genes encoding at least one bile salt metabolism enzyme comprise a polynucleotide sequence having at least 90% sequence identity to the polynucleotide sequence set forth SEQ ID NO: 41 to 43.

[0382] Embodiment 53 is the composition of any one of embodiments 35 through

[0383] 52, wherein the one or more heterologous genes encoding at least one bile salt metabolism enzyme comprise polynucleotide sequence having at least 90% sequence identity to the polynucleotide sequence set forth SEQ ID NO: 44.

[0384] Embodiment 54 is the composition of any one of embodiments 35 through

[0385] 53, wherein the genetically modified Bacteroides thetaiotaomicron further expresses one or more heterologous genes encoding bile acid acyl synthetase for succinyl (BAS-suc); and wherein the BAS-suc is encoded by the one or more heterologous genes comprising an amino acid sequence set forth as SEQ ID NO: 35 or SEQ ID NO:50.

[0386] Embodiment 55 is the composition of any one of embodiments 35 through

[0387] 54, wherein the one or more heterologous genes encoding bile acid acyl synthetase for succinyl (BAS-suc) has at least 90% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 45.

[0388] Embodiment 56 is the composition of any one of embodiments 35 through

[0389] 55, wherein the genetically modified Bacteroides thetaiotaomicron further expresses a BAS-Suc comprising an amino acid sequence set forth as SEQ ID NO: 50, a BSH having an amino acid sequence set forth as SEQ ID NO: 36, or a combination thereof.

[0390] Embodiment 57 is the composition of any one of embodiments 35 through

[0391] 56, wherein the one or more heterologous genes encoding BAS-Suc, BSH, or a combination thereof has at least between 60% to 100% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 46.

[0392] Embodiment 58 is the composition of any one of embodiments 35 through

[0393] 57, wherein the composition is formulated as an agent selected from the group consisting of capsule, pill, yogurt, drink, powder, tablet, suspension, aqueous solution, emulsion, syrup, cream, paste, gel, suppository, ointment, spray, foam, mill, and colloid.

[0394] Embodiment 59 is the composition of any one of embodiments 35 through

[0395] 58, wherein the composition is a pharmaceutical composition, and the carrier is pharmaceutically acceptable carrier.

[0396] Embodiment 60 is the composition of any one of embodiments 35 through

[0397] 59, wherein the concentration of the genetically modified Bacteroides thetaiotaomicron is from about 0.1% to about 100% by weight of the composition.

[0398] Embodiment 61 is the composition of any one of embodiments 35 through

[0399] 60 for use as a medicament.

[0400] Embodiment 62 is the composition of any one of embodiments 35 through

[0401] 61 for use in the treatment of a disease or condition associated with impaired metabolism and / or lipid metabolism.

[0402] Embodiment 63 is the composition of any one of embodiments 35 through

[0403] 62, wherein the disease or condition associated with impaired metabolism is selected from the group consisting of a metabolic disorder, metabolic syndrome, cardiovascular disease, cholestatic disease, hyperlipidemia, dyslipidemia, insulin resistance, type II diabetes, impaired glucose tolerance, obesity, liver disease, NAFLD / MASLD, NASH / MASH, cholestatic disease, or any combination thereof.

[0404] Embodiment 64 is the composition of any one of embodiments 35 through

[0405] 63, wherein the composition is a nutraceutical, supplement, medical food, and / or GRAS designated product.

[0406] Embodiment 65 is a capsule comprising the composition of any one of embodiments 35 through 64, wherein the genetically modified Bacteroides thetaiotaomicron is administered at a concentration of about 1 x 103CFU / animal (or below) to IxlO13CFU / animal (or higher).

[0407] Embodiment 66 is a method of manufacture of a composition comprising a genetically modified bacterial cell and at least one carrier, wherein the genetically modified bacterial cell expresses one or more heterologous genes encoding at least one bile salt metabolism enzyme, wherein the method comprises the steps of: a) Generating a genetically modified bacterial strain, b) Transforming the genetically modified bacterial strain with at least one vector containing one or more heterologous genes encoding at least one bile salt metabolism enzyme; c) Expressing the at least one bile salt metabolism enzyme in the genetically modified bacterial strain; and, d) Preparing the composition comprising the genetically modified bacterial strain and at least one carrier.

[0408] EXPERIMENTAL EXAMPLES

[0409] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0410] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore, specifically point out certain embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0411] Example 1 : Generation of modified bile salt metabolism enzymes

[0412] The current field is recognizing that bile acid (BA) host effects are mediated through receptor-mediated signaling. As such, even if a small fraction of BAs are deconjugated an effect would be expected. These host effects are mediated directly through receptor mediated signaling, and indirectly through modulation of the gut microbiome. These effects include but are not limited to direct activation of FXR, a master regulator of metabolic activity in multiple tissues.

[0413] Thus, there is a need in the field for a precise, targeted therapy, to ensure accurate and safe conversion, rather than a blunt broad and nonspecific approach, that has proved to be unsafe. Additionally, during testing in mice vs humans, studies may need to use targeting specific to mice, including but not limited to MCA muricholic Acid and altering specificity between glyco and tauro conjugates, due to different ratios between the two in different species. Tunability and specificity between these different types of bile salts and bile acids are key for translation from animal models to human subjects.

[0414] To address these problems within the current field a platform technology was developed which has the capability of precise and targeted tunning ability to address each type of BA specifically with different levels of activity, lack of activity, specificity, stability, and more.

[0415] Bacteroides is among the most abundant genera in the human gut microbiome, frequently dominating community profiles in healthy adults (King et al.,

[0416] 2019, PLoS One., 14(9):e0206484). Bacteroides thetaiotaomicron (B. theta) is a well- characterized human commensal that primarily resides in the colon (and is detectable in distal small intestine), where host-microbial conversions of bile acids occur (Aktar et al.,

[0417] 2020, Gut Microbes., 11(6): 1745-1757). Members of Bacteroides encode bile salt hydrolases (BSH / CGH), including in B. theta, and B. theta releases extracellular vesicles that carry a functional BSH (e.g., BT 2086) active in vivo — supporting bile-acid deconjugation activity in the intestinal lumen (Yao et al., 2018, Elife; 7:e37182). Importantly, Bacteroides strains have undergone human testing with favorable safety / tolerability (e.g., B. thetaiotaomicron in a double-blind, placebo-controlled trial in adolescents; B. uniformis strains evaluated for probiotic use), supporting the genus as a viable live biotherapeutic chassis (Hansen et al., 2020, Clin Transl Gastroenterol.; 12(l):e00287). Collectively, B. theta’s abundance, intestinal localization, endogenous BSH activity (including in vesicles), and emerging clinical safety experience make it a strong candidate host for heterologous, high-specificity BSH transgenes engineered to modulate bile-acid pools with precision.

[0418] Clean in-frame deletions of bile-altering enzymes are generated using a counter selectable allelic exchange procedure. 750-basepair regions flanking each gene are amplified by PCR and Hi-Fi DNA Assembly (NEB) is used to clone into pExchange- tdk. Plasmids are verified using Sanger sequencing and electroporated into A. coli S17 and selected for on solid media supplemented with ampicillin to function as a conjugal donor. An approximately equivalent donor-to-recipient ratio is used for conjugation into B. theta Atdk (B. theta with the gene thymidine kinase deleted, which enables counter selection for gene knockouts). Merodiploids are selected after conjugation on solid B. theta media supplemented with gentamicin and erythromycin. A pool of merodiploids is grown in liquid media in the absence of antibiotics to allow for excision of the plasmid from the genome. Successful transformants are selected for solid B. theta media containing FUDR to select against colonies that still contain the plasmid. Single colonies are then verified as successful knockouts using PCR and Sanger sequencing.

[0419] Table 1. Coding sequences used for the modified bile salt hydrolase enzymes and corresponding SEQ ID numbers indicating the corresponding amino-acid and DNA sequences.

[0420] Example 2: Bacteria transformation: Encoding genes in Bacteroides thetaiotaomicron knockout AbshA AbshB AhsdhA

[0421] Table 1 below lists various sequences used for bacterial transfection and subsequent experiments. Plasmids were either commercially available or were synthesized according to order with the appropriate mutations. SEQ ID NO: 40 was used as the backbone plasmid for cloning procedure of coding sequences SEQ ID NOs: 16-30, 41-49, or 51-55.

[0422] E. coli S17-1 strains harboring appropriate plasmids as detailed in Table 1, are grown overnight in LB+lOOug / ml ampicillin. A Bacteroides culture is prepared with overnight anaerobic growth in BHI-S. At mid to late log growth, 200 pL of the appropriate A. coli SI 7-1 cells are spun down, resuspended with 10 pL of a 1 : 10 concentration of the Bacteroides culture, and added to a deep well 96-well plate containing 400 pL of solidified BHI agar per well. After at least 16 hours, the lawn of SI 7-1 and Bacteroides are resuspended in 400 pL of BHI-S by vortex or pipeting, 200 pL of the resuspension is spun down and resuspended in 15 pL BHI-S and several dilutions in BHI-S were made. 3 pL of the resuspension and its dilutions are spotted onto a 120 x 120 mm square petri dish containing BHI agar plus the appropriate antibiotics (200 pg / mL gentamicin and 25 pg / mL erythromycin) and grown anaerobically at 37°C. Colonies present after selection are restruck onto BHI agar plus the appropriate antibiotics (200 pg / mL gentamicin and 25 pg / mL erythromycin) and screened for proper gene insertion using PCR. The engineered gene sequence from verified clones is amplified using PCR and sequence-verified using sanger sequencing.

[0423] Example 3: Testing of genetically modified strains for bile acid degradation

[0424] The following protocol is adapted from Lynch et al., 2023 (Cholestasis impairs gut microbiota development and bile salt hydrolase activity in preterm neonates. Gut Microbes, and is used with slight modifications.

[0425] In vitro characterization of bile transformations by genetically modified B. thetaiotaomicron strains of Example 2 is performed by growing cells in BHI-S media supplemented with 0.5 mM of GCA, GCDCA, TCA, or TCDCA. Cells are grown to stationary phase (shaking at 37 °C), then frozen at -80 °C until further processing. Cells are then thawed, pelleted (5 min at 16,000 x g), and the supernatant is removed to a new microcentrifuge tube. Three volumes of methanol are added, then the mixture is vigorously mixed for 30-60 s and incubated (room temperature, 15 min). Mixtures are centrifuged (5 min, 16,000 x g), the supernatant removed to a clean microcentrifuge tube and dried in a vacuum concentrator. The dried residue is treated with methanol / water / formic acid (50 / 50 / 0.1, all by volume) then vigorously mixed and centrifuged as described above. Supernatants are transferred to polypropylene HPLC vials, capped, and maintained at 4 °C while aliquots (typically 5 pL) are injected onto a reversed phase HPLC column (Cadenza CD-C18, 3.0 pm, 250 x 2 mm, Imtakt) equilibrated in solution A (water / formic acid, 100 / 0.1, vol. / vol.) and eluted (0.2 mb minute- 1) with an increasing concentration of solution B (acetonitrile / formic acid, 100 / 0.1, vol. / vol.); minute / % B: 0 / 30, 45 / 70, 48 / 100, 50 / 30, 67 / 30). The effluent from the column is passed through an electrospray ion source (capillary voltage 42 V, capillary temperature 275 °C, sheath gas flow 15 L min-1, spray voltage 5 kV, and -15 kV conversion dynode with -1.2 kV multipliers) connected to a linear ion trap mass spectrometer (Thermo LTQ) scanning from m / z 95-1000 in the positive ion mode. Spectra are recorded and analyzed with instrument manufacturer supplied software.

[0426] These data demonstrate that native and modified BSH deconjugate bile acids in B. thetaiotaomicron strains and that specific mutations in BSH enhance enzyme activity or substrate specificity. For example, some BSH variants (pPB063 and pPB066, SEQ ID NO: 7 and SEQ ID NO: 10 respectively) demonstrate more versatility as were able to degrade all bile acid substrates. Interestingly, other BSH variants degrade different substrates at different rates demonstrating substrate specificity. Specifically, pPB060 (SEQ ID NO: 4) degraded non-deoxycholate substrates more effectively while and pPB072 (SEQ ID NO: 13) degraded deoxycholate substrates more effectively (Figure 12 through Figure 16). These data demonstrate that specific BSH variants provide enhanced characteristics and unique features that can be utilized to fine-tune bile acid deconjugation and applications thereof.

[0427] Example 4: Test bile acid modification capabilities in a microbiome context The following protocol is adapted from Lynch et al., 2023 and is used with slight modifications. In vitro characterization of bile transformations by genetically modified B. thetaiotaomicron strains as described in Example 2 above, in a microbiome context is performed by growing cells in BHLS media and spiking in IxlO9CFU / ml genetically modified B. thetaiotaomicron and 0.5 mM of GCA, GCDCA, TCA, or TCDCA into fecal material from human donors. Cells and fecal material mixtures are incubated for 12 hours with shaking at 37 °C, then frozen at -80 °C until further processing. Material is then thawed, pelleted (5 min at 16,000 * g), and the supernatant is removed to a new microcentrifuge tube. Three volumes of methanol are added, then the mixture is vigorously mixed for 30-60 s and incubated (room temperature, 15 min). Mixtures are centrifuged (5 min, 16,000 x g), the supernatant removed to a clean microcentrifuge tube and dried in a vacuum concentrator. The dried residue is treated with methanol / water / formic acid (50 / 50 / 0.1, all by volume) then vigorously mixed and centrifuged as described above. Supernatants are transferred to polypropylene HPLC vials, capped, and maintained at 4 °C while aliquots (typically 5 pL) are injected onto a reversed phase HPLC column (Cadenza CD-C18, 3.0 pm, 250 * 2 mm, Imtakt) equilibrated in solution A (water / formic acid, 100 / 0.1, vol. / vol.) and eluted (0.2 mL minute-1) with an increasing concentration of solution B (acetonitrile / formic acid, 100 / 0.1, vol. / vol.); minute / % B: 0 / 30, 45 / 70, 48 / 100, 50 / 30, 67 / 30). The effluent from the column is passed through an electrospray ion source (capillary voltage 42 V, capillary temperature 275 °C, sheath gas flow 15 L min-1, spray voltage 5 kV, and -15 kV conversion dynode with -1.2 kV multipliers) connected to a linear ion trap mass spectrometer (Thermo LTQ) scanning from m / z 95-1000 in the positive ion mode. Spectra are recorded and analyzed with instrument manufacturer supplied software.

[0428] These data imply that treatment with genetically modified Bacteroides thetaiotaomicron mutant BSH-1 or BSH-2, and / or BAS-suc significantly alters the bile acid pool in a native microbiome background, when compared to the control.

[0429] Example 5: In vivo hyperlipidemia / obesity model

[0430] This example establishes transient colonization of genetically modified Bacteroides thetaiotaomicron strains as described in Example 2 above, in Specific- pathogen-free (SPF) mice with and without prior antibiotic treatment. Bile acid species were quantified in cecal material and portal vein to determine if treatment with genetically modified / ?, thetaiotaomicron as described in Example 2 above, i.e. transformed with BSH-1 or BSH-2 enzymes can significantly alter the bile acid pool. This study assessed the ability of genetically modified Bacteroides thetaiotaomicron strains to modify bile acid pool in SPF mice and determined whether genetically modified Bacteroides lhelaiolaomicron. expressing BSH-1 or BSH-2 enhanced enzymatic activity. The study tested BSH-1 and BSH-2 as listed in Table 1. An exemplary treatment grouping is depicted in Table 2.

[0431] Table 2. Treatment Groups

[0432] Timeline and summary of the experiment are as described in Fig. 1, further detailed as follows:

[0433] Protocol:

[0434] Prior to study onset:

[0435] C57B16 wild type female mice aged 4-6 weeks old (Jackson Labs) are randomized into cages of 4 by study group and acclimate to facility for > 1 week.

[0436] Day -7: Mice in antibiotic-treated groups are provided sterilized water with enrofloxacin (0.27 g / L) and ampicillin (1 g / L) ad libitum.

[0437] Day -3: B. theta GFP (hereafter, B. theta WT), B. theta BSH-1, and B. theta BSH-2 on BHI + 25 ug / ml are streaked on erythromycin plates and grown anaerobically at 37C for 48 h. This plate can be stored at 4C and used for the remainder of the experiment.

[0438] Day -1 : liquid cultures of B. theta WT, B. theta BSH-1, and B. theta BSH- 2 in BHLS + 25 ug / ml erythromycin media are inoculated and grown anaerobically at 37C for 18 h.

[0439] Day 0: Antibiotic water in Abx-treated groups is replaced with normal facility water. Cultures are normalized to ~5xl 09CFU / ml (ODeoo = 1 is approximately 5xl08CFU / ml) by washing lx time in sterile deoxygenated PBS and resuspended in appropriate volume of lx PBS.

[0440] For heat-killed material, cell mixture is aliquoted into thin-walled PCR tubes and placed in 70C water bath for 40 min.

[0441] 200 pl heat-killed B. theta, B. theta WT, B. theta BSH-1, or B. theta BSH- 2 are gavage fed per mouse.

[0442] New liquid cultures of B. theta WT, B. theta BSH-1, and B. theta BSH-2 are inoculated in BHI-S + 25 ug / ml erythromycin media and grown anaerobically at 37C for 18 h.

[0443] Day 1 : Cultures are normalized to ~5xl09CFU / ml (ODeoo = 1 is approximately 5xl08CFU / ml) by washing lx time in sterile deoxygenated PBS and resuspended in appropriate volume of lx PBS.

[0444] For heat-killed material, cell mixture is aliquoted into thin-walled PCR tubes and placed in 70C water bath for 40 min.

[0445] 200 pl heat-killed B. theta, B. theta WT, B. theta BSH-1, or B. theta Eng BSH-2 are gavage fed per mouse.

[0446] New liquid cultures of B. theta WT, B. theta BSH-1, and B. theta Eng BSH-2 are inoculated in BHI-S + 25 ug / ml erythromycin media and grown anaerobically at 37C for 18 h.

[0447] Day 2: Cultures are normalized to ~5xl09CFU / ml (ODeoo = 1 is approximately 5xl08CFU / ml) by washing lx time in sterile deoxygenated PBS and resuspending in appropriate volume of lx PBS.

[0448] For heat-killed material, aliquot cell mixture into thin-walled PCR tubes and place in 70C water bath for 40 min.

[0449] 200 pl heat-killed B. theta, B. theta WT, B. theta E'SEE , or B. theta Eng BSH-2 are gavage fed per mouse.

[0450] Day 7:Mice are sacrificed and the following samples are collected * Distal small intestinal contents are collected, mass for normalization is measured, and 750 pl of Zymo DNA / RNA shield buffer are added for RT storage. Cecal contents are being collected and split between two tubes and mass for normalization is measured. 750 pl Zymo DNA / RNA shield buffer is added to one tube for RT storage and the other tube is flash-frozen and stored at -80C.

[0451] Blood from portal vein is collected and allowed to clot in SST Vacutainer tubes (BD) on ice. supernatant is centrifuged, removed and flash frozen and stored at - 80C.

[0452] Analyses: qPCR*

[0453] Metabolomics: frozen cecal and serum samples on dry ice is sent to Metabolon for bile acid measurement using the total bile analysis pipeline.

[0454] *CFU enumeration: Colonization is measured using CFU counts instead of qPCR given the introduced antibiotic resistance in these strains. A starting point would be to plate distal SI and cecal contents from a non-colonized SPF mouse on BBE (Bacteroides bile esculin) agar with 25 pg / ml erythromycin and 30 pg / ml vancomycin to determine what the background growth would be.

[0455] This study determined if treatment with genetically modified Bacteroides thetaiotaomicron expressing mutated BSH-1 or BSH-2 significantly impacted weight gain in mice fed a high-fat diet. Weight gain over the 12-week study was the primary endpoint. This study also determined if treatment with B. theta expressing BSH-1 or BSH-2 significantly impacted biological readouts related to bile acids and disease outcomes. This study established whether treatment with engineered B. theta can modify host biology over the course of long-term dosing. This study also determined if treatment with B. theta expressing BSH-1 or BSH-2 significantly impacted biological readouts related to ex-vivo assays such as monitoring GLP-1 mRNA and protein levels in plasma, serum, or tissue samples.

[0456] Groups:

[0457] Final treatment groups (Abx, dosing, etc.) was informed by colonization study prior to the onset of this efficacy study.

[0458] An exemplary treatment grouping is depicted in Table 3.

[0459] Table 3: Treatment groups

[0460] A preliminary study on colonization and bile acid modification was used to inform whether BSH-1 or BSH-2 was selected for this study. For the remainder of the protocol, BSH-1 is used as a placeholder for BSH-1 or BSH-2. The study is testing BSH- 1 and BSH-2 variants as listed in Table 1.

[0461] Timeline and summary of the experiment are as described in Fig. 2, further detailed as follows:

[0462] Protocol:

[0463] Prior to study onset: a. C57B16 wild type mice aged 4-6 weeks old are acclimated to facility for > 1 week. b. Mice are caged randomly in groups of 5 by study group such that the average starting weight of each group is equal. c. Each mouse is ear-tagged with an ID.

[0464] For all B. theta dosing:

[0465] • Three days prior to first dosing, B. theta GFP (hereafter, B. theta WT) and B. theta BSH-1 on BHI + 25 ug / ml are streaked on erythromycin plates and grown anaerobically at 37C for 48 h. These plates can be stored at 4C and used for up to two weeks before an additional fresh plate is needed.

[0466] • The day prior to each dosing, liquid cultures of B. theta WT and B. theta BSH-1 are inoculated in BHI-S + 25 ug / ml erythromycin media and grown anaerobically at 37C for 18 h.

[0467] • The day of dosing, cultures are normalized to ~5xl09CFU / ml (ODeoo = 1 is approximately 5xl08CFU / ml) by washing lx time in sterile deoxygenated PBS and resuspending in appropriate volume of lx PBS.

[0468] • For heat-killed material, cell mixture is aliquoted into thin-walled PCR tubes and placed in 70C water bath for 40 min.

[0469] • 200 pl heat-killed B. theta, B. theta WT or B. theta BSH-1 are gavage fed per mouse. Weekly measurements:

[0470] • Each week, individual mouse weights is measure and food consumption is cage- leveled.

[0471] Day -7:

[0472] 1. Mice in antibiotic-treated groups are provided sterilized water with enrofloxacin (0.27 g / L) and ampicillin (1 g / L) ad libitum.

[0473] Day O '.

[0474] • Antibiotic water in Abx-treated groups is replaced with normal facility water.

[0475] • 200 pl heat-killed B. theta freshly-prepared B. theta WT, or freshly-prepared B. theta BSH-1 are gavage fed for -IxlO9CFU / mouse.

[0476] Day 1:

[0477] • 200 pl heat-killed B. theta, freshly-prepared B. theta WT, or freshly-prepared B. theta BSH-1 are gavage fed for -IxlO9CFU / mouse.

[0478] Day 2:

[0479] • 200 pl heat-killed B. theta, freshly-prepared B. theta WT, or freshly-prepared B. theta BSH-1 are gavage fed for -IxlO9CFU / mouse.

[0480] Day 4:

[0481] • 200 pl heat-killed B. theta, freshly-prepared B. theta WT, or freshly-prepared B. theta BSH-1 are gavage fed for -IxlO9CFU / mouse.

[0482] Day 7:

[0483] • Mice are provided with high-fat diet (fat (40% kcal), high fructose (20% kcal), and cholesterol (2% kcal)).

[0484] Days 7-84:

[0485] • Performing 2 doses / week by gavaging 200 pl heat-killed B. theta, freshly prepared B. theta WT, or freshly prepared B. theta BSH-1 for - IxlO9CFU / mouse.

[0486] • Individual mouse weights are measured, and food consumption is cage-level once per week.

[0487] • Cages are changed once per week, taking care to prevent cross-contamination. Day 83: • Mice are fasting the day prior to sacrifice to enable secondary endpoint measurements.

[0488] Day 84:

[0489] • Mice are sacrificed and tissues are collected for secondary endpoints as outlined in the “analyses” section below.

[0490] Analyses:

[0491] The following tissues are collected: Serum, fecal pellets, colon tissue, small bowel tissue, liver, fat (subcutaneous, gonadal, and mesenteric).

[0492] The following experiments are performed on collected tissue. All assays are performed using commercially available and validated kits in biological duplicates or sent to an external vendor for analysis.

[0493] • Endpoint colonization in SI and colon using qPCR

[0494] • Cecal contents and serum will be sent for bile acid measurements with Metab ol on.

[0495] • H / E staining for liver

[0496] • Oil red o staining for liver

[0497] • TG content for liver

[0498] • Total cholesterol, HDL, LDL, TG content for serum.

[0499] • Glucose for serum

[0500] • Insulin for serum

[0501] • Samples will be stored for future analysis which may include PCR for FXR, TGR5, fatty acid oxidation.

[0502] This data analysis implies that treatment with genetically modified Bacteroides ihetaiolaomicron mutant BSH-1 or BSH-2 significantly impacts weight gain in mice fed a high-fat diet, when compared to the control.

[0503] Administration of Bacteroides thetaiotaomicron engineered to express bile salt hydrolases (BSHs) resulted in enhanced deconjugation of bile acids in the intestinal lumen. Increased liberation of free bile acids augmented signaling through the bile acid receptor TGR5 on enteroendocrine L-cells. Consistent with prior reports that free bile acids such as deoxycholic acid (DCA) and lithocholic acid (LCA) are potent TGR5 agonists (Maruyama et al., 2002, J. Biol. Chem.), colonization with engineered B. thetaiotaomicron led to increased TGR5 activation and stimulated secretion of the incretin hormone GLP-1. Elevated GLP-1 secretion is well established to enhance glucose-dependent insulin release and lower postprandial glycemia (Thomas et al., 2009, Nature; Watanabe et al., 2006, Nature. 439(7075):484-9).

[0504] In murine models colonized with B. thetaiotaomicron expressing engineered BSHs, significantly elevated GLP-1 levels were observed following nutrient challenge compared to animals colonized with control strains lacking engineered BSH activity. These findings are consistent with prior demonstrations that engineered BSH- expressing bacteria remodel bile acid pools and improve host metabolic signaling (Joyce et al., 2014, Proc Natl Acad Sci. 111(20):7421-6; Yao et al., 2018, Cell Metab).

[0505] Example 6: In vivo MASH protocol

[0506] Efficacy of the different B. theta strains as described in Example 2 above, on MASH disease progression in murine models is tested as follows:

[0507] Briefly, 8-week-old SPF male mice are fed a choline-deficient amino acid- defined and high fat diet (CDAA-HFD) for 8 weeks. Mice are treated with control and test strains twice per week for the final three weeks. For biochemical analysis, hepatic and plasma triglyceride and cholesterol is determined with assay kits following the manufacturer’s instructions. ALT and AST levels are assessed by using commercial ALT and AST assay kits. For histological analysis of liver, after mice sacrifice, liver tissues of mice are preserved in 10% formalin. Formalin-fixed paraffin-embedded liver tissue slides are subjected to hematoxylin and eosin (H&E) and Sirius red staining to assess lipid accumulation, inflammation severity and liver fibrosis degree, respectively. The frozen liver sections are stained with oil red O for visualizing lipid droplets. All the processes are performed according to standard protocols followed by microscopic examination. The histology scores are evaluated according to a reported MAFLD scoring system and Sirius red positive areas are measured by ImageJ.

[0508] Various analysis performed on collected tissues are conducted according to previous Example 6, such as bile-acid analysis performed on collected fecal pellets. This data analysis implies that treatment with genetically modified Bacteroides thetaiotaomicron expressing BAS-suc significantly alters the bile acid pool in a native microbiome background, when compared to the control. This implies that treatment with genetically modified Bacteroides thetaiotaomicron expressing BAS-suc significantly alleviate symptoms and biomarkers of MAFLD / MASH, when compared to the control.

[0509] Example 7: In-vivo cholestatic disease model

[0510] A BALB / c.Mdr2- / - mouse model of PSC is used to test efficacy of engineered strains. 3 -week-old mice are treated with control and modified strains twice per week for 9 weeks. Liver cirrhosis is used as a primary readout.

[0511] Various analysis performed on collected tissues are conducted according to previous Example 6, such as bile-acid analysis performed on collected fecal pellets.

[0512] This data analysis implies that treatment with genetically modified Bacteroides thetaiotaomicron mutant BSH-1 or BSH-2 significantly alters the bile acid pool in a native microbiome background, when compared to the control. This implies that treatment with genetically modified Bacteroides thetaiotaomicron mutant BSH-1 or BSH-2 significantly improve disease outcomes in a murine model of cholestatic disease, when compared to the control.

[0513] Example 8: Screening for activity of BSH variants in E. coli.

[0514] The aim of this protocol is to describe the complete set-up of experiments from E. coli cultivation to detection of the product after enzymatic reaction of cell lysate with substrate.

[0515] BSH expression

[0516] Pre-culture Microtiter plates were prepared with 145 pL LB medium including 50 pg / mL Kanamycin. The cryogenic cultures of the replicates (BSH2 in inhouse strain E. coli BL21(DE3) derivative) were thawed. After resuspension, each well was inoculated with 5 pL of thawed glycerol stock. The MTP, containing 150 pL inoculated medium was sealed with a lid and incubated at 37 °C for 16 h under shaking at 900 rpm. Main culture

[0517] To determine the growth of the cells, 5 pL of pre-culture were diluted 1 :40 in 195 pL LB medium and the absorption at 600 nm was measured. For the main culture, 495 pL of LB medium incl. 50 pg / mL Kanamycin were added to each well of the deepwell plate. 5 pL of the pre-culture were added to the medium to reach a final OD600 ~ 0.05. The inoculated medium was incubated at 37 °C under shaking at 900 rpm and 80 % humidity until the bacterial cultures reached an OD600 ~ 1.0, therefore the OD600 was measured during cultivation. Once OD600 ~ 1.0 was reached, the cells were induced with 5 pL of a 10 mM IPTG stock to a final concentration of 100 pM IPTG. After induction, the DWPs were sealed with a gas-permeable foil. Expression took place at 37 °C under shaking at 900 rpm and 80 % humidity for 20 h.

[0518] Harvest

[0519] After 20 h of expression the cells were harvested by centrifugation of the plates at 4,000 g for 10 min at 4 °C. Afterwards the supernatant was discarded. The cell pellet was frozen at -20 °C for at least 1 h.

[0520] BSH assay Lysis

[0521] To obtain BSH, the cells need to be lysed. The cell pellet after expression was stored at -20 °C. After at least 1 h at -20 °C the cell pellet was thawed at 37 °C. 1 mg / mL Lysozyme solution was prepared. Lysozyme was either resuspended in pH 5 buffer or pH 7 buffer, depending on the pH of the BSH assay reaction. Per cell pellet 160 pL of the lysis buffer were used. The cell pellet was resuspended thoroughly and incubated at 37 °C for 30 min under shaking at 900 rpm. After 30 min the lysed cells were centrifuged at 4,000 g for 10 min at 4 °C. The supernatant (SLF) contains the soluble BSH. Until the start of the BSH assay the SLF was kept on ice.

[0522] BSH assay preparation

[0523] The respective buffers (sodium acetate buffer pH 5 and sodium phosphate buffer pH 7) were pre-warmed in a water bath to 37 °C. The F-botom MTP was also preincubated at 37 °C. The 2 mM TCA solution in dH2O was freshly prepared on the day of the assay. The 2 mM TCDCA solution, 2 mM GCA solution and 2 mM GCDCA solution were prepared freshly on the day of the assay by 1 : 10 dilution in dH2O from a 20 mM TCDCA / GCA / GCDCA stock in 100 % DMSO. The solutions were protected from light.

[0524] Enzyme dilution

[0525] The SLF was diluted in sodium acetate buffer pH 5 or sodium phosphate buffer pH 7. These steps were always proceeded on ice.

[0526] Preparation of deproteination of samples

[0527] To prepare for the deproteination, the ultrafiltration plate was washed with 150 pL of the respective buffer. The wash step took place at 4 °C with 4,000 g for 5 min. The remaining retentate was discarded and the plate was centrifuged dry at 4 °C with 4,000 g for 5 min.

[0528] BSH assay

[0529] The pre-warmed MTP was kept at 37 °C and 20 pL of enzyme dilution were transferred into the MTP. 115 pL of pre-warmed buffer either pH 5 or pH 7 were added to the 20 pL enzyme dilution. Afterwards, 15 pL of 2 mM substrate solution were added to the buffer / enzyme dilution solution. The BSH assay final concentrations were as follows: 200 pM substrate, 180 mM buffer in pH 5 or pH 7. The BSH assay reactions were incubated for 10 min under shaking at 900 rpm at 37 °C.

[0530] Stopping the reaction / deproteination

[0531] After 10 min BSH assay reaction at 37 °C, 120 pL of reaction mix were transferred immediately with a 96-channel pipette to the prewashed ultrafiltration plate. The transferred reaction mix was centrifuged at 4.000 g for 5 min at 4 °C. Filtrate was stored at 4 °C for subsequent use or immediately frozen at -80 °C for longer storage.

[0532] Taurine detection assay

[0533] Preparation of the Taurine assay

[0534] The samples from the BSH assay were thawed. From the taurine assay kit taurine standards and taurine assay buffer were thawed at RT. The residual components were thawed on ice. Standards were prepared in duplicates with 0 pM, 15.6 pM, 31.3 pM, 62.5 pM, 125 pM, 250 pM, 500 pM and 1000 pM. The reaction mix was prepared according to the manual, see Table 4. An exemplary enzymatic reaction for taurine formation with the disclosed BSH enzymes is depicted in Fig. 3.

[0535] Table 4: Reaction mix for taurine assay kit

[0536] Preparation of the samples

[0537] On one hand, 50 pL of deproteinated sample from BSH assay at pH 7 were transferred into an MTP. On the other hand, 33 pL of deproteinated sample from BSH assay at pH 5 were transferred into an MTP and 17 pL of 200 mM TRIS / HC1 buffer, pH 9 were added to these samples. Addition of pH 9 buffer to the pH 5 samples was necessary to reach a pH of ~ 7. All samples were performed as duplicates. One duplicate was directly measured with the taurine kit, whereas the other duplicate was quenched in order to determine the sample background.

[0538] Taurine assay

[0539] 50 pL of the standard were transferred into an MTP. The reaction was started with 50 pL of reaction mix. Incubation was performed under shaking at 25 °C for 30 min. The reaction was stopped with 50 pL of stop solution and 50 pL of developing solution. For detection of sample background, the reaction was quenched with 50 pL of quenching solution and 50 pL of developing solution and incubated under shaking for 3 min at 25 °C. The detection of absorbance (415 nm) was performed in a plate reader. The absorption of sample background (=quenched) was subtracted from the absorption of the sample, which was stopped with stop solution.

[0540] Glycine detection assay

[0541] Preparation of the Glycine assay

[0542] The samples from the BSH assay were thawed. From the glycine assay kit glycine standards, and glycine assay buffer were thawed at RT. The residual components were thawed on ice. Standards were prepared in duplicates with 0 pM, 1.56 pM, 3.13 pM, 6.25 pM, 12.5 pM, 25.0 pM, 50.0 pM and 100.0 pM. The reaction mix was prepared according to the manual, see Table 5. An exemplary enzymatic reaction for glycine formation with the disclosed BSH enzymes is depicted in Fig. 4.

[0543] Table 5: Reaction mix for glycine assay kit

[0544] Preparation of the samples

[0545] The samples with pH 7 were diluted 1 :3 in assay buffer. Samples with pH 5 were diluted 2:3 in buffer at pH 9 and then diluted further 1 :2 in assay buffer.

[0546] Glycine assay

[0547] 50 pL of standard were transferred into a black MTP. 50 pL of 1 :3 diluted samples were transferred into a black MTP. The reaction was started with 50 pL of reaction mix. Incubation was performed protected from light under shaking at 900 rpm at 37 °C for 30-60 min. The fluorescence was measured at Ex / Em 560 / 587 after 60 min.

[0548] Results

[0549] Total protein was run on an SDS-PAGE gel and stained with Coomassie Blue to visualize total production of BSH as depicted in Fig. 5.

[0550] As can been seen in the stained band corresponding to the L67Y BSH1 variant, L67Y variant shows higher protein production compared to the WT enzyme.

[0551] Example 9: Treatment of non-alcoholic fatty liver disease

[0552] A subject suffering from non-alcoholic fatty liver disease (NAFLD / MASLD) is administered a therapeutically effective amount of the disclosed compositions, e.g., pharmaceutical compositions, prebiotics, or probiotics. The compositions are administered by any route suitable for the treatment needs of the subject, preferably, oral or intrarectal routes. Signs and symptoms of the NAFLD / MASLD are measured and assessed using suitable tools, checks, tests, and / or questionnaire. Results

[0553] Surprisingly, signs and symptoms of NAFLD / MASLD are reduced by the administration of the compositions, as compared to an untreated subject.

[0554] Example 10: Treatment of non-alcoholic steatohepatitis

[0555] The presence and severity of nonalcoholic steatohepatitis is associated with specific changes in circulating bile acids (Puri et al., 2018, Hepatology. 67(2):534- 548). Previous methods have had a blunt broad and nonspecific approach that has proved to be unsafe. For example, Obeticholicacid (OCA / Ocaliva) a synthetic bile acid (obeticholic acid) that is an FXR agonist, meaning it activates the farnesoid X receptor to regulate bile acid metabolism and downstream liver function. Ocaliva is not an antagonist; its therapeutic effects in diseases like primary biliary cholangitis are achieved by stimulating FXR activity rather than blocking it. OCA proved Purpose Bio’s biology works, where in clinical trials for both MASH and PBC efficacy was shown. However, the side effect profile was not acceptable and it was withdrawn from the trials. OCA being a “sledgehammer” approach and a systemic delivery, targeting FXR directly, is different than the present approach as the present study demonstrates a non-systemic delivery, targeting the enterohepatic circulation of bile acids (BA) as a whole, restoring native proper function and balance, by fine tuning the specific levels of primary and secondary BA, and avoiding a direct target on FXR alone.

[0556] A subject suffering from non-alcoholic steatohepatitis (NASH / MASH) is administered a therapeutically effective amount of the disclosed compositions, e.g., pharmaceutical compositions, prebiotics, or probiotics. The compositions are administered by any route suitable for the treatment needs of the subject, e.g., oral or intrarectal routes, at any suitable dosing regimen, and over an appropriate period of time. Signs and symptoms of NASH / MASH are measured and assessed using suitable tools, checks, tests, and / or questionnaire.

[0557] Results

[0558] Surprisingly signs and symptoms of NASH / MASH are reduced by the administration of the compositions of the invention, as compared to an untreated subject. Example 1 1 : Validation of engineered strains for bile acid deconjugation activity.

[0559] Recent studies have shown that expression of Bile Salt Hydrolase (BSH) protein in Bacteroides thetaiotaomicron can impact circulating lipid species related to various cardiometabolic diseases. Enzyme engineering has been performed to change the activity and properties of BSH and has validated the in vitro results using this model.

[0560] Six to eight week old C57BL / 6 mice were be divided into the following 6 groups (6 mice per group) and received gavage of the indicated material on study days 0, 1, and 2. For bacterial groups, 200 ul of 5xlO10CFU / ml bacteria resuspended in saline were gavaged. For 1 week prior to dosing (study day -7 to study day 0), mice were provided enrofloxacin (0.27 g / L) and ampicillin (1 g / L) ad libitum in drinking water, which was replaced with normal water upon study initiation (day 0). Mice were fed a normal chow diet.

[0561] 1. Saline

[0562] 2. Bacteroides thetaiotaomicron WT

[0563] 3. Bacteroides thetaiotaomicron BSH1

[0564] 4. Bacteroides thetaiotaomicron BSH2

[0565] 5. Bacteroides thetaiotaomicron BSH3

[0566] 6. Bacteroides thetaiotaomicron BSH4

[0567] At the end of the treatment period, mice fasted for 4 hours and blood and cecal material were collected to analyze bile acid species.

[0568] Table 6

[0569] Table 7

[0570] Mice were fed special water (+ / - antibiotics). Lab personnel were responsible for monitoring the food amount and changing the food weekly.

[0571] Mice fasted for 4 hours prior to blood collection and euthanasia.

[0572] Example 12: Primary sclerosing cholangitis to evaluate the impact of Bacteroides thetaiotaomicron BSH strains.

[0573] Recent studies have shown that expression of Bile Salt Hydrolase (BSH) protein in Bacteroides thetaiotaomicron can impact circulating lipid species related to various cardiometabolic diseases. This model tested the impact of these strains on outcomes in primary sclerosing cholangitis (PSC).

[0574] Four week old mice were divided into the following 4 groups (12 mice per group) and received gavage of the indicated material three days per week (MWF) for 6 weeks. For bacterial groups, 200 ul of 5xlO10CFU / ml bacteria resuspended in saline were gavaged. For 1 week prior to dosing (study day -7 to study day 0), mice were provided enrofloxacin (0.27 g / L) and ampicillin (1 g / L) ad libitum in drinking water, which was replaced with normal water upon study initiation (day 0). Mice were fed normal chow for the entirety of the study.

[0575] 1. Saline

[0576] 2. Bacteroides thetaiotaomicron WT

[0577] 3. Bacteroides thetaiotaomicron BSH1

[0578] 4. Bacteroides thetaiotaomicron BSH 2

[0579] At the end of the treatment period, mice fasted for 4 hours and blood and cecal material were collected to analyze bile acid and various lipid species. Liver tissue was also collected for ex vivo analysis.

[0580] Table 8

[0581] Table 9

[0582] Mice were fed antibiotics in water. Mice fasted for 4 hours prior to blood collection and euthanasia.

[0583] Example 13: High fat diet to evaluate the impact of Bacteroides thetaiotaomicron BSH strains.

[0584] In another example, Cholestyramine (CMA) is a therapy that binds the entire BA pool indiscriminately and was shown to cause unacceptable GI side effects. Additionally, this approach also results in the loss of critical signaling benefits of the BA in the host health. Moreover, between 20%-50% of Hyperlipidemia / Statin patients, either refuse or discontinue Statin therapy due to side effects, and this increases their risk for cardiovascular events by up to 50%. Recent studies have shown that expression of Bile Salt Hydrolase (BSH) protein in Bacteroides thetaiotaomicron can impact circulating lipid species related to various cardiometabolic diseases. Enzyme engineering was performed to change the activity and properties of BSH and the in vitro results were validated using this model. Specifically, the impact of these engineered strains on weight gain, circulating lipid species, and liver endpoints was tested.

[0585] Six to eight week old C57BL / 6 mice were divided into the following 6 groups (12 mice per group) and received gavage of the indicated material three days per week (MWF) for 14 weeks. For bacterial groups, 200 ul of 5xlO10CFU / ml bacteria resuspended in saline were gavaged. For 1 week prior to dosing (study day -7 to study day 0), mice were provided enrofloxacin (0.27 g / L) and ampicillin (1 g / L) ad libitum in drinking water, which was replaced with normal water upon study initiation (day 0). Mice were fed a high fat high cholesterol diet (Research Diets D09100310) to promote obesity traits. Weight was measured once per week for the entire course of the study.

[0586] 1. Saline

[0587] 2. Saline (weeks 1-6), Bacteroides thetaiotaomicron BSH1 (weeks 7-14)

[0588] 3. Bacteroides thetaiotaomicron WT

[0589] 4. Bacteroides thetaiotaomicron BSH 1

[0590] 5. Bacteroides thetaiotaomicron BSH2

[0591] 6. Bacteroides thetaiotaomicron BSH1 + Bacteroides uniformis

[0592] At the end of the treatment period, mice fasted for 4 hours and blood and cecal material were collected to analyze bile acid and various lipid species. Liver and white adipose tissue were also collected for ex vivo analysis.

[0593] Table 10

[0594] Table 11

[0595] Mice were fed a special diet and antibiotics in water. Lab personnel were responsible for monitoring the food amount and changing the food weekly.

[0596] Mice fasted for 4 hours prior to blood collection and euthanasia.

[0597] Example 14: Secondary Bile Acid Protocol

[0598] Secondary bile acids are generated when gut microbes chemically modify primary bile acids. Secondary bile acids act as potent signaling metabolites that influence many aspects of human health including metabolic health, immune regulation, and gut integrity.

[0599] Assessing secondary bile acid production utilizing 7alpha-dehydroxylator samples for DCA production

[0600] 1. Preculture strains (B. thetaiotaomicron with engineered BSH and 7alpha- dehydroxylator strain ex, Clostridium scindens) anaerobically in BHI-S media overnight separately.

[0601] 2. Inoculate each strain to an OD600=0.1 and combine at a ratio of 2: 1 to 10: 1 (B. theta: C. scindens)

[0602] 3. Spike cultures with ,125uM to ,5uM GCA.

[0603] 4. Incubate anaerobically at 37C for 24 to 72 hours and sample at multiple time points from 0 to 24-72 hours.

[0604] 5. Prepare samples utilizing LCMS protocol and run LCMS to assess DCA production.

[0605] Assessing secondary bile acid production utilizing fecal samples for DCA production

[0606] 1. Inoculate genetically modified B. thetaiotaomicron anaerobically in BHI-S media overnight. 2. Dilute into fresh BHI-S media to OD600=0.1 and add 0.125uM to 0.5uM GCA.

[0607] 3. Incubate anaerobically at 37C for 24 to 72 hours and sample at multiple time points from 0 to 24-72 hours.

[0608] 4. Centrifuge culture at 3000 x g for 10 minutes at 4C to pellet cells.

[0609] 5. Filter supernatant through 0.22 um sterile fdter in anaerobic conditions.

[0610] 6. Weigh fecal samples and add pre-reduced PBS to make a 10% w / v fecal slurry. Homogenize until slurry is uniform.

[0611] 7. Mix fdtered supernatant at a 1 : 1 to 1 :5 ratio with 10% fecal slurry in anaerobic conditions. Incubate at 37C for 24 to 72 hours. Collect at time points from 0 to 24-72 hours.

[0612] 8. Exact samples using LCMS protocol and run LCMS to assess DC A production.

[0613] LCMS Protocol

[0614] 1. Centrifuge media samples for 5 min at 3000 x g at 4 °C to remove any cells.

[0615] 2. Collect the supernatant in a new centrifuge tube on ice.

[0616] 3. Centrifuge the medium at 16,000xg for 5 min at 4 °C to remove any cell debris. If desired, transfer the content to a new centrifuge tube and store at -80 °C.

[0617] 4. Prepare Eppendorf tubes with 500 pl ice-cold 80% MeOH / 20% water and place on ice.

[0618] 5. Add 20 pl of clarified medium from step 3.

[0619] 6. Vortex for 30 sec

[0620] 7. Incubate for 1 hr at -80 °C.

[0621] 8. Place on ice to warm up. Vortex briefly.

[0622] 9. Spin for 10 min at 16,000 xg at 4 °C.

[0623] 10. Transfer 450 ul of the supernatant into a glass vial

[0624] 11. Dry in a Genevac evaporator or in a speed vac without applying heat. Choose an appropriate drying time and remove the samples promptly when the program is finished.

[0625] Example 15: Coexpression of native BAS-suc with native BSH and native BAS-suc with modified BSHs Bile acid succinylation is a microbial transformation that alters bile-acid physicochemistry and signaling. In Bacteroides uniformis, bile acid acyl synthetasesuccinyl (BAS-suc) catalyzes the succinylation of unconjugated bile acids (e.g., CA —> 3- succinyl-CA (3-sucCA)) using succinate as acyl donor. To demonstrate portability and function in a non-native host, Bacteroides thetaiotaomicron (VPI-5482) was engineered in two formats:

[0626] 1. BAS-suc-only construct, and

[0627] 2. BAS-suc + BSH construct (where BSH provides unconjugated substrates from conjugated bile acids).

[0628] Exemplary studies (LC-MS workflow and ranges). Anaerobic cultures (OD600 -0.05-0.1 inoculum) were grown in defined medium supplemented with:

[0629] • For BAS-suc-only: unconjugated CA, CDCA, or DCA (each -10-500 pM) plus sodium succinate (-1-50 mM).

[0630] • For BAS-suc + BSH: conjugated GCA (-10-500 pM) plus sodium succinate (~1- 50 mM).

[0631] Aliquots were taken over 0-48(-72) h (e.g., 0, 2, 6, 12, 24, 48 h), supernatants were protein-precipitated with cold methanol (3-5 x v / v), clarified, and analyzed by LC-MS( / MS) in negative ESI on a C18 column (water / acetonitrile + 0.1% formic acid or ammonium acetate). Targeted acquisition monitored parent— >product ions of 3-sucCA, 3-sucCDCA, and 3-sucDCA alongside their unconjugated precursors; external calibration curves (e.g., 1-1000 nM) and stable-isotope internal standards supported quantitation.

[0632] Results (representative).

[0633] • BAS-suc-only: time-dependent formation of 3-sucCA, 3- sucCDCA, 3-sucDCA from respective unconjugated substrates, with substrate preference for CA (highest conversion), consistent with native B. uniformis BAS- suc selectivity.

[0634] • BAS-suc + BSH: in media containing GCA, BSH produced CA, which was succinylated by BAS-suc; LC-MS showed progressive 3-sucCA accumulation with concomitant GCA depletion and CA turnover. Conclusion. BAS-suc is functionally expressed in B. thetaiotaomicron, and both (i) BAS-suc-only and (ii) BAS-suc + BSH constructs generate succinylated bile acid products detectable and quantifiable by LC-MS, reproducing the native enzyme’s substrate selectivity in a heterologous chassis.

[0635] Example 16: pH stability

[0636] To evaluate the robustness of bile salt hydrolase (BSH) and bile acid succinyl synthetase (BAS-suc) enzymes under physiologically relevant conditions, constructs were assessed for activity and stability at pH 5.0 and pH 7.0, reflecting the range encountered in the human colon.

[0637] Methods. Bacteroides thetaiotaomicron (VPI-5482) strains were generated in multiple formats:

[0638] 1. Native BAS-suc constructs, in which the native BAS-suc enzyme from Bacteroides uniformis was heterologously expressed in B. thetaiotaomicron.

[0639] 2. Engineered BSH constructs, in which BSH enzymes were modified by amino acid substitutions, deletions, or insertions to improve activity or stability.

[0640] 3. Native BSH constructs, in which BSH enzymes were expressed without sequence modification.

[0641] Crude lysates from each construct were normalized for protein concentration and incubated in sodium phosphate buffer (pH 5.0 or 7.0) containing 0.125-0.5 pM glycocholic acid (GCA) as substrate and, where appropriate, sodium succinate as co-substrate. Reactions were performed anaerobically at 37 °C for 1-24 hours, then quenched with ice-cold methanol. Products were quantified by LC-MS( / MS) through detection of cholic acid (CA) and 3 -succinyl derivatives, using external calibration curves (1—1000 nM) and isotope-labeled internal standards.

[0642] Results. Both native BAS-suc and BSH constructs (native and engineered) were active at pH 5.0 and 7.0. The engineered BSH variants retained >70-90% of maximal activity at pH 5.0, whereas native BSHs showed partial loss of activity under acidic conditions. The native BAS-suc enzyme retained robust activity across the tested range and generated succinylated derivatives of cholic acid and related substrates. Conclusion. The disclosure encompasses (i) native BAS-suc enzymes heterologously expressed in non-native hosts, and (ii) BSH enzymes, both native and engineered, expressed in thetaiotaomicron or alternative commensals. These enzymes function effectively under colonic pH conditions, enabling oral, non-systemic delivery of bile acid-modifying activity with enhanced stability compared to wild-type BSHs alone.

[0643] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMSWhat is claimed is:

1. A modified bile salt hydrolase (BSH) having at least 80% homology to an amino acid sequence set forth as SEQ ID NO: 34, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33 and comprising one or more substitutions relative to SEQ ID NO: 34, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33.

2. The modified BSH of claim 1, wherein the modified BSH comprises at least 90% homology to an amino acid sequence set forth as SEQ ID NO: 34, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33 and comprising one or more substitutions relative to SEQ ID NO: 34, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33.

3. The modified BSH of claim 1, wherein the modified BSH comprises an amino acid sequence set forth as SEQ ID NO: 7, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 4, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

4. The modified BSH of claim 1, wherein the modified BSH comprises at least one amino acid substitution at position L67, KI 95, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 31 or 33.

5. The modified BSH of claim 1, wherein the modified BSH comprises a combination of at least two amino acid substitutions at any of the positions L67, K195, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 31 or 33.

6. The modified BSH of claim 1, wherein the modified BSH comprises a combination of multiple amino acid substitutions at any of the positions L67, K195,R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 31 or 33.

7. The modified BSH of claim 1, wherein the modified BSH comprises at least one amino acid substitution at position Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 32.

8. The modified BSH of claim 1, wherein the modified BSH comprises at least one amino acid substitution at position Y34, H47, 157, L127, K189, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34.

9. The modified BSH of claim 1, wherein the modified BSH comprises at least two amino acid substitutions at any of the positions Y34, H47, 157, L127, KI 89, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34.

10. The modified BSH of claim 1, wherein the modified BSH comprises a combination of multiple amino acid substitutions at any of the positions Y34, H47, 157, L127, K189, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID NO: 34.

11. The modified BSH of claim 1, wherein the modified BSH is expressed in a modified bacterial cell.

12. The modified BSH of claim 11, wherein the modified bacterial cell is Bacteroides thetaiotaomicron .

13. An isolated oligonucleotide having at least 80% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 41 to 44.

14. The isolated oligonucleotide of claim 13, wherein the isolated oligonucleotide comprises at least 90% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 41 to 44.

15. The isolated oligonucleotide of claim 13, wherein the isolated oligonucleotide comprises a polynucleotide sequence set forth as SEQ ID NO: 16 to 30 or SEQ ID NO: 51 to 55.

16. An expression vector comprising the isolated oligonucleotide of any of any one of claims 13-15 operably linked to at least one promotor.

17. A composition comprising an isolated oligonucleotide of any one of claims 13-15, an expression vector of claim 16, a modified BSH of any one of claims 1-12, or any combination thereof, and at least one carrier.

18. A composition of claim 17, further comprising a bile acid acyl synthetase for succinyl (BAS-suc) comprising an amino acid sequence set forth as SEQ ID NO: 35, SEQ ID NO:50, or a homologue thereof.

19. The composition of claim 17 or 18 for use as a medicament.

20. The composition of claim 17 or 18, wherein the composition is a nutraceutical, supplement, medical food, and / or GRAS designated products.

21. The composition of claim 17 or 18 for use in the treatment of a disease or condition associated with impaired metabolism.

22. A method of treating a disease or condition associated with impaired metabolism, comprising administering to the subject in need an effective amount of the composition of claim 17 or 18.

23. A method of treating a condition associated with impaired metabolism and / or lipid metabolism, comprising administering to a subject in need of such treatment an effective amount of the composition of claim 17 or 18.

24. A method of alleviating at least one symptom of a disease or condition associated with impaired metabolism comprising administering to a subject afflicted with said disease or condition an effective amount of the composition of claim 17 or 18.

25. The method of claim 24, wherein the disease or condition is associated with impaired lipid metabolism.

26. A method of preventing and / or delaying an onset and / or or preventing and / or slowing progression of a disease or condition associated with impaired metabolism comprising administering to a subject an amount of the composition of claim 17 or 18 effective to prevent or delay progression of a disease or condition associated with impaired metabolism.

27. The method of claim 26, wherein the disease or condition is associated with impaired lipid metabolism.

28. The method of any one of claims 22-27, wherein the condition associated with impaired metabolism is selected from the group consisting of a metabolic disorder, metabolic syndrome, cardiovascular disease, cholestatic disease, hyperlipidemia, dyslipidemia, insulin resistance, type II diabetes, impaired glucose tolerance, obesity, liver disease, NAFLD / MASLD, NASH / MASH, cholestatic disease, or any combination thereof.

29. The method of any one of claims 22-27, wherein the composition is administered orally, rectally, intravenously, intranasally, ocularly, subcutaneously, transdermally, or transmucosally.

30. A method for increasing bile acid deconjugation, wherein the method comprises administering the composition of claim 17 or 18.

31. The method of claim 30, wherein the method targets a specific species of BAs.

32. The method of claim 30, wherein the method increases deconjugation of circulating bile acids.

33. A pharmaceutical composition comprising a modified bile salt hydrolase (BSH) enzyme, formulated in a capsule or pill or any other delivery method, wherein the composition is effective to modulate bile acid metabolism in a subject.

34. The pharmaceutical composition of claim 33, wherein the modified BSH is administered at a concentration of about 0.1% to 100% by weight of the composition.

35. A composition comprising a genetically modified Bacteroides thetaiotaomicron and at least one carrier, wherein the genetically modified Bacteroides thetaiotaomicron expresses one or more heterologous genes encoding at least one bile salt metabolism enzyme; and wherein the bile salt metabolism enzyme encoded by the one or more heterologous genes is characterized by having enhanced enzymatic activity, lower or lack of activity, and / or substrate specificity and / or stability.

36. The composition of claim 35, wherein the genetically modified Bacteroides thetaiotaomicron bears a knockout mutation in at least one of the genes encoding bile acid or bile salt metabolism enzyme.

37. The composition of claim 35, wherein the at least one bile salt metabolism enzyme is a bile salt hydrolase (BSH).

38. The composition of claim 35, wherein the BSH is a native Christensenella minuta BSH.

39. The composition of claim 35, wherein the at least one bile salt metabolism enzyme comprises an amino acid sequence set forth as SEQ ID NO: 34, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33.

40. The composition of claim 35, wherein the at least one bile salt metabolism enzyme comprises an amino acid sequence set forth as SEQ ID NO: 7, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 4, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

41. The composition of claim 35, wherein the genetically modified Bacteroides thetaiotaomicron expresses a combination of two or more heterologous genes encoding two or more bile salt metabolism enzymes, and wherein the two or more bile salt metabolism enzymes are characterized by having enhanced enzymatic activity, lower or lack of activity, and / or substrate specificity and / or stability, and, wherein the two or more bile salt metabolism enzymes, independently of each other, are characterized by having an amino acid sequence set forth as SEQ ID NO: 34, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, EQ ID NO: 7, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 4, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

42. The composition of claim 37, wherein the BSH comprises at least one amino acid substitution at position L67, K195, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 31 or 33.

43. The composition of claim 37, wherein the BSH comprises a combination of at least two amino acid substitutions at any of the positions L67, KI 95, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 31 or 33.

44. The composition of claim 37, wherein the BSH comprises a combination of multiple amino acid substitutions at any of the positions L67, K195, R297, or Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 31 or 33.

45. The composition of claim 37, wherein the BSH comprises at least one amino acid substitution at position Q328, when compared to the amino acid sequence set forth as SEQ ID NO: 32.

46. The composition of claim 37, wherein the BSH comprises at least one amino acid substitution at position Y34, H47, 157, L127, KI 89, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID: 34.

47. The composition of claim 37, wherein the BSH comprises a combination of at least two amino acid substitutions at any of the positions Y34, H47, 157, L127, K189, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID: 34.

48. The composition of claim 37, wherein the BSH comprises a combination of multiple amino acid substitutions at any of the positions Y34, H47, 157, L127, K189, N292, or R323, when compared to the amino acid sequence set forth as SEQ ID: 34.

49. The composition of claim 35, wherein the one or more heterologous genes encoding at least one bile salt metabolism enzyme comprise a polynucleotide sequence having at least 80% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 16 to SEQ ID NO:30 or SEQ ID NO: 51 to SEQ ID NO: 55.

50. The composition of claim 35, wherein the one or more heterologous genes encoding at least one bile salt metabolism enzyme comprise a polynucleotide sequence having at least 90% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 16 to SEQ ID NO:30 or SEQ ID NO: 51 to SEQ ID NO: 55.51 . The composition of claim 35, wherein the one or more heterologous genes encoding at least one bile salt metabolism enzyme comprise a polynucleotide sequence having between 80% to 99% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 16 to SEQ ID NO:30 or SEQ ID NO: 51 to SEQ ID NO: 55.

52. The composition of claim 35, wherein the one or more heterologous genes encoding at least one bile salt metabolism enzyme comprise a polynucleotide sequence having at least 90% sequence identity to the polynucleotide sequence set forth SEQ ID NO: 41 to 43.

53. The composition of claim 35, wherein the one or more heterologous genes encoding at least one bile salt metabolism enzyme comprise polynucleotide sequence having at least 90% sequence identity to the polynucleotide sequence set forth SEQ ID NO: 44.

54. The composition of any one of claims 35-53, wherein the genetically modified Bacteroides thetaiotaomicron further expresses one or more heterologous genes encoding bile acid acyl synthetase for succinyl (BAS-suc); and wherein the BAS-suc is encoded by the one or more heterologous genes comprising an amino acid sequence set forth as SEQ ID NO: 35 or SEQ ID NO: 5055. The composition of claim 54, wherein the one or more heterologous genes encoding bile acid acyl synthetase for succinyl (BAS-suc) has at least 90% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 45.

56. The composition of any one of claims 35-53, wherein the genetically modified Bacteroides thetaiotaomicron further expresses a BAS-Suc comprising an amino acid sequence set forth as SEQ ID NO: 50, a BSH having an amino acid sequence set forth as SEQ ID NO: 36, or a combination thereof.

57. The composition of claim 56, wherein the one or more heterologous genes encoding BAS-Suc, BSH, or a combination thereof has at least between 60% to 100% sequence identity to the polynucleotide sequence set forth as SEQ ID NO: 46.

58. The composition of any one of claims 35-57, wherein the composition is formulated as an agent selected from the group consisting of capsule, pill, yogurt, drink, powder, tablet, suspension, aqueous solution, emulsion, syrup, cream, paste, gel, suppository, ointment, spray, foam, mill, and colloid.

59. The composition of any one of claims 35-57, wherein the composition is a pharmaceutical composition, and the carrier is pharmaceutically acceptable carrier.

60. The composition of any one of claims 35-57, wherein the concentration of the genetically modified Bacteroides thetaiotaomicron is from about 0.1% to about 100% by weight of the composition.

61. The composition of any one of claims 35-57 for use as a medicament.

62. The composition of any one of claims 35-57 for use in the treatment of a disease or condition associated with impaired metabolism and / or lipid metabolism.

63. The composition for use according to claim 61, wherein the disease or condition associated with impaired metabolism is selected from the group consisting of a metabolic disorder, metabolic syndrome, cardiovascular disease, cholestatic disease, hyperlipidemia, dyslipidemia, insulin resistance, type II diabetes, impaired glucose tolerance, obesity, liver disease, NAFLD / MASLD, NASH / MASH, cholestatic disease, or any combination thereof.

64. The composition of claim 35, wherein the composition is a nutraceutical, supplement, medical food, and / or GRAS designated product.

65. A capsule comprising the composition of claim 35, wherein the genetically modified Bacteroides thetaiotaomicron is administered at a concentration of about 1 x 103CFU / animal (or below) to IxlO13CFU / animal (or higher).

66. A method of manufacture of a composition comprising a genetically modified bacterial cell and at least one carrier, wherein the genetically modified bacterial cell expresses one or more heterologous genes encoding at least one bile salt metabolism enzyme, wherein the method comprises the steps of a) Generating a genetically modified bacterial strain, b) Transforming the genetically modified bacterial strain with at least one vector containing one or more heterologous genes encoding at least one bile salt metabolism enzyme; c) Expressing the at least one bile salt metabolism enzyme in the genetically modified bacterial strain; and, d) Preparing the composition comprising the genetically modified bacterial strain and at least one carrier.

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