Modified bile salt hydrolases and methods of use thereof

Abstract

Disclosed are modified Cristinia parvata bile salt hydrolase (BSH) enzymes, prebiotics comprising the same, engineered bacterial cells comprising the modified BSH enzymes, probiotics comprising the engineered bacterial cells, methods of treating diseases and conditions using compositions comprising the modified enzymes, and food products.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 493,219, filed March 30, 2023, the entire contents of which are incorporated herein by reference.

[0003] sequence list

[0004] This application is submitted with an ordered list as a sequence list XML file named “179136_00009.xml”, which is 116,308 bytes in size and was created on March 29, 2024. The sequence list was submitted electronically via the Patent Center and is incorporated herein by reference in its entirety. Technical Field

[0005] The technical field of this disclosure relates to modified bile salt hydrolases, compositions comprising said modified enzymes, and methods of using said enzymes. Background Technology

[0006] Bile salt hydrolases (BSH) catalyze the deconjugation of bile acids, playing a crucial role, particularly in dietary fat absorption and microbiome regulation. Furthermore, BSH enzyme function is associated with diseases in humans and animals. Therefore, there is a need in this field for modified BSH enzymes. Summary of the Invention

[0007] Modified Christensenella minuta bile salt hydrolase

[0008] In one aspect of this disclosure, a modified *Christsenium spp.* bile salt hydrolase (BSH) enzyme is provided. In some embodiments, the modified enzyme is at least 90% identical to SEQ ID NO: 1 and contains one or more amino acid substitutions at positions C2, Y8, Y34, N46, H47, Y56, I57, Y64, P81, K90, F99, L127, S136, Y176, K189, S206, R207, A221, N292, or R323 relative to SEQ ID NO: 1. In some implementations, one or more alternatives include Y8K, Y8T, Y8Q, Y8V, Y8T, Y34N, Y34S, N46K, H47S, H47N, Y56A, Y56M, Y56T, Y56L, Y56V, I57V, Y64S, Y64F, P81A, K90E, F99Y, L127I, L127V, S136A, S136T, Y176K, K189P, K189N, S206G, R207Q, A221P, A221L, N292S, N292R, N292H, R323P, R323T, R323E, R323L, or R323Q. In some embodiments, one or more substitutions include Y8V, Y34K, I57V, Y64S, P81A, K90E, L127I, S136A, K189P, K189N, S206G, R207Q, N292S, R323L, or R323E. In some embodiments, one or more substitutions include I57V. In some embodiments, one or more substitutions include Y64S. In some embodiments, one or more substitutions include S136A. In some embodiments, one or more substitutions include K189P. In some embodiments, one or more substitutions include Y8V, P81A, S206G, and R207Q. In some embodiments, one or more substitutions include Y34K, I57V, L127I, and N292S. In some embodiments, one or more substitutions include Y56T, I57V, L127I, and N292S. In some embodiments, one or more substitutions include K90E, L127I, N292S, and R323E. In some embodiments, one or more substitutions include Y34N, N46K, H47N, Y64F, Y176K, and A221L. In some embodiments, one or more substitutions include Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L. In some embodiments, one or more substitutions include C2S. In some embodiments, the modified enzyme comprises or consists of one of SEQ ID NO: 2-47.In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 5, and wherein the modified enzyme has a substitution I57V relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 5. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 6, and wherein the modified enzyme has a substitution Y64S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 6. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 8, and wherein the modified enzyme has a substitution S136A relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 9, and wherein the modified enzyme has a substituted K189P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 36, and wherein the modified enzyme has substituted Y8V, P81A, S206G, and R207Q relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 36. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 37, and wherein the modified enzyme has substituted Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 37. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 41, and wherein the modified enzyme has substitutions of Y56T, I57V, L127I, and N292S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 41. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 42, and wherein the modified enzyme has substitutions of K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 42.In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 43, and wherein the modified enzyme has substitutions of Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 43. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 44, and wherein the modified enzyme has substitutions of Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 44. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 45, and wherein the modified enzyme has substitutions of Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1. Optionally, the modified enzyme comprises or is composed of SEQ ID NO: 45. In some embodiments, the modified enzyme exhibits increased activity compared to wild-type C. minuta BSH at approximately pH 5. In some embodiments, the modified enzyme exhibits increased activity compared to wild-type C. minuta BSH at approximately pH 7. In some embodiments, the modified C. minuta bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, wherein the modified BSH enzyme has at least one substitution modification relative to SEQ ID NO: 1.

[0009] In one aspect of this disclosure, a modified BSH enzyme is provided. In some embodiments, the modified bile salt hydrolase (BSH) enzyme comprises amino acid substitutions at the following positions relative to SEQ ID NO: 1: I5V, Y8R, T9S, D11H, H12T, Y13F, R16M, L18Y, L20A, E21S, F22S, Y24E, N25G, T27K, V28I, T29V, V30I, T31V, K33R, Y34K, F37L, H38K, F39L, R40S, K43E, L45I, N46S, H47N, M54I, Y56K, I57V, V58I, D60N, F61Y, Y64F, Y65K, D66M, T68C, S74A. G77A, L78I, N79S, D82G, N83I, D85S, K87G, V89P, K90T, E91P, Y93K, D94E, I96V, F99Y, F101L, W104Y, Q108R, A110G, S113D, R11 6L, I117E, E120K, Q121T, I122L, L124I, L127V, N128D, E131A, L133H, S136K, L138V, H139K, W140Y, Q145K, R146E, D147K, V151L , S153Q, F161Y, V165T, N170G, F174Y, M178L, K189N, E190K, T195K, A197S, A198P, E199S, L200I, E201K, Q204R, Y205I, R207S, A211N, L218Y, S220D, A221L, R223K, V225I, K226R, A228S, T230V, K231R, M232L, S234A, E240Y, S241D, S243Q, I244L, S245M, G2 52D, E255R, Q256N, Q257V, R258P, C260T, E265G, E269L, I270R, I272L, S274Q, S275T, C277I, N278D, K281R, Y284L, T287R, E290N, N292T, Y300N, E302C, N303D, D305N, N307D, T308K, S311E, Y312F, M315V, K316T, Q318P, N321Y, Y322E, R323L, N324K, and Y325K.In some embodiments, the modified bile salt hydrolase (BSH) enzyme comprises amino acid substitutions at the following positions relative to SEQ ID NO: 1: I5V, Y8K, T9S, D11S, H12S, Y13W, R16M, L18Y, L20A, E21S, F22S, Y24E, T27E, V28I, T29V, V30I, T31V, K33R, Y34K, F37L, H38K, F39L, R40S, K43T, L45I, N46D, H47E, Y49N, M54I, Y56E, V58R, D60N, L 63I, Y64F, Y65K, D66M, G77A, L78V, N79S, F80L, D82G, N83I, D85S, K87G, V89K, K90T, Y93K, D94E, F99Y, F10 1L, W104Y, Q108R, C109A, A110S, S113E, R116L, I117K, Q121N, I122L, L124I, L127V, N128D, L133R, S136K, L138V, H139R, W140F, S143A, Q145K, R146D, D147K, S148A, S153Q, F161Y, V165T, N170G, T173L, F174Y, M178L, K189N, E190A, T195K, A198P, E199N, L 200I, E201K, Q204R, Y205I, R207A, M209D, A211N, L218K, S220D, A221L, R2 23K, V225I, K226R, A228T, T230V, K231R, M232L, S234A, S239T, S241D, S24 3Q, I244L, S245M, G252D, E255R, Q256D, Q257V, R258P, C260T, H262W, E265G, E269L, I270R, I272L, S274Q, S275V, C276A, C277I, K281T, I283K, Y284L, T287R, E290N, N292S, T295N, A296M, Y300N, E302C, N303D, D305N, G306S, N307D, T308K, Y312F, M315Q, Q318L, Q319D, N321K, Y322E, R323L, and Y325K. In some embodiments, the modified bile salt hydrolase (BSH) enzyme comprises or consists of one of SEQ ID NO: 46 or 47.

[0010] Polynucleotides

[0011] In one aspect of this disclosure, a polynucleotide is provided. In some embodiments, the polynucleotide comprises a sequence encoding a modified *Kristensenia pyrrhizina* bile salt hydrolase (BSH) enzyme, said modified *Kristensenia pyrrhizina* bile salt hydrolase (BSH) enzyme being at least 90% identical to SEQ ID NO: 1, and comprising one or more amino acid substitutions at positions C2, Y8, Y34, N46, H47, Y56, I57, Y64, P81, K90, F99, L127, S136, Y176, K189, S206, R207, A221, N292, or R323 relative to SEQ ID NO: 1. In some embodiments, the modified enzyme is at least 90% identical to SEQ ID NO: 1 and contains one or more amino acid substitutions at positions C2, Y8, Y34, N46, H47, Y56, I57, Y64, P81, K90, F99, L127, S136, Y176, K189, S206, R207, A221, N292, or R323 relative to SEQ ID NO: 1. In some implementations, one or more alternatives include Y8K, Y8T, Y8Q, Y8V, Y8T, Y34N, Y34S, N46K, H47S, H47N, Y56A, Y56M, Y56T, Y56L, Y56V, I57V, Y64S, Y64F, P81A, K90E, F99Y, L127I, L127V, S136A, S136T, Y176K, K189P, K189N, S206G, R207Q, A221P, A221L, N292S, N292R, N292H, R323P, R323T, R323E, R323L, or R323Q. In some embodiments, one or more substitutions include Y8V, Y34K, I57V, Y64S, P81A, K90E, L127I, S136A, K189P, K189N, S206G, R207Q, N292S, R323L, or R323E. In some embodiments, one or more substitutions include I57V. In some embodiments, one or more substitutions include Y64S. In some embodiments, one or more substitutions include S136A. In some embodiments, one or more substitutions include K189P. In some embodiments, one or more substitutions include Y8V, P81A, S206G, and R207Q. In some embodiments, one or more substitutions include Y34K, I57V, L127I, and N292S. In some embodiments, one or more substitutions include Y56T, I57V, L127I, and N292S. In some implementations, one or more alternatives include K90E, L127I, N292S, and R323E.In some embodiments, one or more substitutions include Y34N, N46K, H47N, Y64F, Y176K, and A221L. In some embodiments, one or more substitutions include Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L. In some embodiments, one or more substitutions include C2S. In some embodiments, the modified enzyme comprises or consists of one of SEQ ID NO: 2-47. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 5, and wherein the modified enzyme has the substitution I57V relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 5. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 6, and wherein the modified enzyme has a substituted Y64S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 6. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 8, and wherein the modified enzyme has a substituted S136A relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 9, and wherein the modified enzyme has a substituted K189P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 36, and wherein the modified enzyme has substitutions of Y8V, P81A, S206G, and R207Q relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 36. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 37, and wherein the modified enzyme has substitutions of Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 37.In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 41, and wherein the modified enzyme has substitutions of Y56T, I57V, L127I, and N292S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 41. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 42, and wherein the modified enzyme has substitutions of K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 42. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 43, and wherein the modified enzyme has substitutions of Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 43. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 44, and wherein the modified enzyme has substitutions of Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 44. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 45, and wherein the modified enzyme has substitutions of Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1. Optionally, the modified enzyme comprises or is composed of SEQ ID NO: 45. In some embodiments, the modified enzyme exhibits increased activity compared to wild-type *Kristensenia spp.* BSH at approximately pH 5. In some embodiments, the modified enzyme exhibits increased activity compared to wild-type *Kristensenia spp.* BSH at approximately pH 7. In some embodiments, the modified *Kristensenia spp.* bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, wherein the modified BSH enzyme has at least one substitution modification relative to SEQ ID NO: 1. In some embodiments, the polynucleotide further comprises one or more regulatory elements, wherein the regulatory elements are operatively linked to a sequence encoding a modified enzyme. In some embodiments, the one or more regulatory elements comprise a promoter and / or an enhancer.In some embodiments, the polynucleotide further comprises a selection marker. In some embodiments, one or more regulatory elements are promoters, and the promoters are constitutive promoters. In some embodiments, one or more regulatory elements are promoters, and the promoters are inducible promoters. In some embodiments, the promoter may be induced by environmental conditions or in response to hypoxic or anaerobic conditions. In some embodiments, the polynucleotide comprises one of SEQ ID NO: 48-79.

[0012] prebiotics

[0013] In one aspect of this disclosure, a prebiotic is provided. In some embodiments, the prebiotic comprises a modified *Kristensenia pyrrhizina* bile salt hydrolase (BSH) enzyme that is at least 90% identical to SEQ ID NO: 1 and contains one or more amino acid substitutions at positions C2, Y8, Y34, N46, H47, Y56, I57, Y64, P81, K90, F99, L127, S136, Y176, K189, S206, R207, A221, N292, or R323 relative to SEQ ID NO: 1. In some implementations, one or more alternatives include Y8K, Y8T, Y8Q, Y8V, Y8T, Y34N, Y34S, N46K, H47S, H47N, Y56A, Y56M, Y56T, Y56L, Y56V, I57V, Y64S, Y64F, P81A, K90E, F99Y, L127I, L127V, S136A, S136T, Y176K, K189P, K189N, S206G, R207Q, A221P, A221L, N292S, N292R, N292H, R323P, R323T, R323E, R323L, or R323Q. In some embodiments, one or more substitutions include Y8V, Y34K, I57V, Y64S, P81A, K90E, L127I, S136A, K189P, K189N, S206G, R207Q, N292S, R323L, or R323E. In some embodiments, one or more substitutions include I57V. In some embodiments, one or more substitutions include Y64S. In some embodiments, one or more substitutions include S136A. In some embodiments, one or more substitutions include K189P. In some embodiments, one or more substitutions include Y8V, P81A, S206G, and R207Q. In some embodiments, one or more substitutions include Y34K, I57V, L127I, and N292S. In some embodiments, one or more substitutions include Y56T, I57V, L127I, and N292S. In some embodiments, one or more substitutions include K90E, L127I, N292S, and R323E. In some embodiments, one or more substitutions include Y34N, N46K, H47N, Y64F, Y176K, and A221L. In some embodiments, one or more substitutions include Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L. In some embodiments, one or more substitutions include C2S.In some embodiments, the modified enzyme comprises or consists of one of SEQ ID NO: 2-47. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 5, and wherein the modified enzyme has a substitution I57V relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 5. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 6, and wherein the modified enzyme has a substitution Y64S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 6. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 8, and wherein the modified enzyme has a substitution S136A relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 9, and wherein the modified enzyme has a substituted K189P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 36, and wherein the modified enzyme has substituted Y8V, P81A, S206G, and R207Q relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 36. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 37, and wherein the modified enzyme has substituted Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 37. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 41, and wherein the modified enzyme has substitutions of Y56T, I57V, L127I and N292S relative to SEQ ID NO: 1, optionally wherein the modified enzyme comprises or is composed of SEQ ID NO: 41.In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 42, and wherein the modified enzyme has substitutions of K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 42. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 43, and wherein the modified enzyme has substitutions of Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 43. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 44, and wherein the modified enzyme has substitutions of Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 44. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 45, and wherein the modified enzyme has substitutions of Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 45. In some embodiments, the modified enzyme exhibits increased activity compared to wild-type *Kristensenia spp.* BSH at approximately pH 5. In some embodiments, the modified enzyme exhibits increased activity compared to wild-type *Kristensenia spp.* BSH at approximately pH 7. In some embodiments, the modified *Kristensenia spp.* bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, wherein the modified BSH enzyme has at least one substitution modification relative to SEQ ID NO: 1.

[0014] Pharmaceutical compositions containing modified BSH enzymes

[0015] In one aspect of this disclosure, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical composition comprises a modified *Christsenium spp.* bile salt hydrolase (BSH) enzyme that is at least 90% identical to SEQ ID NO: 1 and contains one or more amino acid substitutions at positions C2, Y8, Y34, N46, H47, Y56, I57, Y64, P81, K90, F99, L127, S136, Y176, K189, S206, R207, A221, N292, or R323 relative to SEQ ID NO: 1. In some implementations, one or more alternatives include Y8K, Y8T, Y8Q, Y8V, Y8T, Y34N, Y34S, N46K, H47S, H47N, Y56A, Y56M, Y56T, Y56L, Y56V, I57V, Y64S, Y64F, P81A, K90E, F99Y, L127I, L127V, S136A, S136T, Y176K, K189P, K189N, S206G, R207Q, A221P, A221L, N292S, N292R, N292H, R323P, R323T, R323E, R323L, or R323Q. In some embodiments, one or more substitutions include Y8V, Y34K, I57V, Y64S, P81A, K90E, L127I, S136A, K189P, K189N, S206G, R207Q, N292S, R323L, or R323E. In some embodiments, one or more substitutions include I57V. In some embodiments, one or more substitutions include Y64S. In some embodiments, one or more substitutions include S136A. In some embodiments, one or more substitutions include K189P. In some embodiments, one or more substitutions include Y8V, P81A, S206G, and R207Q. In some embodiments, one or more substitutions include Y34K, I57V, L127I, and N292S. In some embodiments, one or more substitutions include Y56T, I57V, L127I, and N292S. In some embodiments, one or more substitutions include K90E, L127I, N292S, and R323E. In some embodiments, one or more substitutions include Y34N, N46K, H47N, Y64F, Y176K, and A221L. In some embodiments, one or more substitutions include Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L. In some embodiments, one or more substitutions include C2S.In some embodiments, the modified enzyme comprises or consists of one of SEQ ID NO: 2-47. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 5, and wherein the modified enzyme has a substitution I57V relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 5. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 6, and wherein the modified enzyme has a substitution Y64S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 6. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 8, and wherein the modified enzyme has a substitution S136A relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 9, and wherein the modified enzyme has a substituted K189P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 36, and wherein the modified enzyme has substituted Y8V, P81A, S206G, and R207Q relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 36. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 37, and wherein the modified enzyme has substituted Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 37. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 41, and wherein the modified enzyme has substitutions of Y56T, I57V, L127I and N292S relative to SEQ ID NO: 1, optionally wherein the modified enzyme comprises or is composed of SEQ ID NO: 41.In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 42, and wherein the modified enzyme has substitutions of K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 42. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 43, and wherein the modified enzyme has substitutions of Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 43. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 44, and wherein the modified enzyme has substitutions of Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 44. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 45, and wherein the modified enzyme has substitutions of Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 45. In some embodiments, the modified enzyme exhibits increased activity compared to wild-type *Kristensenia spp.* BSH at approximately pH 5. In some embodiments, the modified enzyme exhibits increased activity compared to wild-type *Kristensenia spp.* BSH at approximately pH 7. In some embodiments, the modified *Kristensenia spp.* bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, wherein the modified BSH enzyme has at least one substitution modification relative to SEQ ID NO: 1.

[0016] engineered bacterial cells

[0017] In one aspect of this disclosure, engineered bacterial cells are provided. In some embodiments, the engineered bacterial cells comprise a polynucleotide containing a sequence encoding a modified *Kristensenia pyrrhizina* bile salt hydrolase (BSH) enzyme, said modified *Kristensenia pyrrhizina* bile salt hydrolase (BSH) enzyme being at least 90% identical to SEQ ID NO: 1, and containing one or more amino acid substitutions at positions C2, Y8, Y34, N46, H47, Y56, I57, Y64, P81, K90, F99, L127, S136, Y176, K189, S206, R207, A221, N292, or R323 relative to SEQ ID NO: 1. In some embodiments, the modified enzyme is at least 90% identical to SEQ ID NO: 1, and contains one or more amino acid substitutions at positions C2, Y8, Y34, N46, H47, Y56, I57, Y64, P81, K90, F99, L127, S136, Y176, K189, S206, R207, A221, N292, or R323 relative to SEQ ID NO: 1.Position 1 contains one or more amino acid substitutions at C2, Y8, Y34, N46, H47, Y56, I57, Y64, P81, K90, F99, L127, S136, Y176, K189, S206, R207, A221, N292, or R323. In some implementations, one or more alternatives include Y8K, Y8T, Y8Q, Y8V, Y8T, Y34N, Y34S, N46K, H47S, H47N, Y56A, Y56M, Y56T, Y56L, Y56V, I57V, Y64S, Y64F, P81A, K90E, F99Y, L127I, L127V, S136A, S136T, Y176K, K189P, K189N, S206G, R207Q, A221P, A221L, N292S, N292R, N292H, R323P, R323T, R323E, R323L, or R323Q. In some embodiments, one or more substitutions include Y8V, Y34K, I57V, Y64S, P81A, K90E, L127I, S136A, K189P, K189N, S206G, R207Q, N292S, R323L, or R323E. In some embodiments, one or more substitutions include I57V. In some embodiments, one or more substitutions include Y64S. In some embodiments, one or more substitutions include S136A. In some embodiments, one or more substitutions include K189P. In some embodiments, one or more substitutions include Y8V, P81A, S206G, and R207Q. In some embodiments, one or more substitutions include Y34K, I57V, L127I, and N292S. In some embodiments, one or more substitutions include Y56T, I57V, L127I, and N292S. In some embodiments, one or more substitutions include K90E, L127I, N292S, and R323E. In some embodiments, one or more substitutions include Y34N, N46K, H47N, Y64F, Y176K, and A221L. In some embodiments, one or more substitutions include Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L. In some embodiments, one or more substitutions include C2S. In some embodiments, the modified enzyme comprises or consists of one of SEQ ID NO: 2-47. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 5, and wherein the modified enzyme has the substitution I57V relative to SEQ ID NO: 1; optionally, the modified enzyme comprises SEQ ID NO: 5 or consists of SEQ ID NO: 1.5. Composition. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 6, and wherein the modified enzyme has a substituted Y64S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 6. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 8, and wherein the modified enzyme has a substituted S136A relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 8. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 9, and wherein the modified enzyme has a substituted K189P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 9. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 36, and wherein the modified enzyme has substitutions of Y8V, P81A, S206G, and R207Q relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 36. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 37, and wherein the modified enzyme has substitutions of Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 37. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 41, and wherein the modified enzyme has substitutions of Y56T, I57V, L127I, and N292S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 41. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 42, and wherein the modified enzyme has substitutions of K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 42. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 43, and wherein the modified enzyme has substitutions of Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1. Optionally, the modified enzyme comprises SEQ ID NO: 43.SEQ ID NO: 43 or is composed of SEQ ID NO: 43. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 44, and wherein the modified enzyme has substitutions of Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1; optionally, the modified enzyme comprises SEQ ID NO: 44 or is composed of SEQ ID NO: 44. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 45, and wherein the modified enzyme has substitutions of Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1; optionally, the modified enzyme comprises SEQ ID NO: 45 or is composed of SEQ ID NO: 45. In some embodiments, the modified enzyme exhibits increased activity compared to wild-type *Kristensenia spp.* BSH at approximately pH 5. In some embodiments, the modified enzyme exhibits increased activity compared to wild-type *Kristensenia spp.* BSH at approximately pH 7. In some embodiments, the modified *Kristensenia spp.* bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, wherein the modified BSH enzyme has at least one substitution modification relative to SEQ ID NO: 1. In some embodiments, the polynucleotide further comprises one or more regulatory elements, wherein the regulatory elements are operatively linked to the sequence encoding the modified enzyme. In some embodiments, one or more regulatory elements comprise a promoter and / or an enhancer. In some embodiments, the polynucleotide further comprises a selection marker. In some embodiments, one or more regulatory elements are promoters, and the promoters are constitutive promoters. In some embodiments, one or more regulatory elements are promoters, and the promoters are inducible promoters. In some embodiments, the promoter may be induced by environmental conditions or in response to hypoxic or anaerobic conditions. In some embodiments, the polynucleotide comprises one of SEQ ID NO: 48-79. In some embodiments, the expression of the polynucleotide is operatively linked to a foreign promoter not found in the native Christensenella pyrenoidosa genome. In some embodiments, the engineered bacterial cells are selected from the group consisting of: certain species of the genus *Acidaminococcus*, certain species of the genus *Actinomyces*, *Akkermansia muciniphila*, certain species of the genus *Allobaculum*, and certain species of the genus *Anaerococcus*.spp.), certain species of the genus *Anaerostipes*, certain species of the genus *Bacteroides*, other species of the genus *Bacteroides*, *Bacteroides acidifacicens*, *Bacteroides coprophilus*, *Bacteroides fragilis*, *Bacteroides ovatus*, *Bacteroides uniformis*, certain species of the genus *Barnesiellaceae*, *Bifidobacteriuma adolescentis*, other species of the genus *Bifidobacterium*, certain species of the genus *Bifidobacterium*, certain species of the genus *Bilophila*, *Blautiaobeum*, *Blautia producta*, other species of the genus *Blautia*. Other species, some species of *Blautia* spp., some species of *Bulleidia* spp., some species of *Catenibacterium* spp., some species of *Chrisenella* spp., some species of *Citrobacter* spp., some species of *Clostridiaceae* spp., other species of *Clostridiales*, some species of *Clostridiales* spp., *Clostridium perfringens*, some species of *Clostridium* spp., other species of *Clostridium* spp., *Collinsella aerofaciens*, some species of *Collinsella* spp., *Collinsella fecal* *Stercoris*, *Coprococcus catus*, certain species of *Coprococcus* spp., certain species of *Coriobacteriaceaes* spp., certain species of *Desulfovibrionaceae* spp., and certain species of *Dialister* spp.Dorea formicigenerans, certain species of Dorea, other species of Dorea, Eggerthella lenta, other species of Enterobacteriaceae, certain species of Enterobacteriaceae, certain species of Enterococcus, certain species of Erysipelotrichaceae, Eubacterium biforme, Eubacterium dolichum, certain species of Eubacterium, Faecalibacterium prausnitzii, certain species of Fusobacterium, and certain species of Gemellaceae. Haemophilus parainfluenzae, other species of Haemophilus, certain species of Helicobacter, other species of Helicobacter (Lachnospiraceae), certain species of Lachnospiracea (Lachnospiraceaespp.), Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus zeae, certain species of Lactobacillus, certain species of Lactobacillus (Lactobacillus spp.), certain species of Lactobacillaceae, certain species of Lactococcus, certain species of Leuconostocaceae, certain species of Megamonas, and certain species of Megasphaera. spp.), certain species of the genus *Methanobrevibacter*, *Mitsuokella multacida*, certain species of the genus *Mitsuokella*, *Mucispirillum schaedleri*, and certain species of the genus *Odoribacter*spp.), certain species of the genus *Oscillospira*, *Parabacteroides distasonis*, certain species of the genus *Parabacteroides*, certain species of the genus *Parabacteroides*, certain species of the genus *Paraprevotella*, certain species of the family *Paraprevotellaceae*, certain species of the genus *Parvimonas*, certain species of the genus *Pediococcus*, other species of the genus *Pediococcus*, certain species of the genus *Peptococcus*, certain species of the genus *Peptoniphilus*, anaerobic *Peptostreptococcus anaerobius*, other species of the genus *Peptostreptococcus*, certain species of the genus *Phascolarctobacterium* Prevotella copri, certain species of Prevotella, Prevotella stercorea, Prevotellaceae, certain species of Proteus, certain species of Rikenellaceae, Roseburia faecis, certain species of Roseburia, other species of Ruminococcus, certain species of Ruminococcus, Ruminococcus bromii, Ruminococcus gnavus, certain species of Ruminococcus, other species of Ruminococcus, Ruminococcus twitch Torques), certain species of Slackia, certain species of S24-7, certain species of SMB53, Streptococcus anginosus, Streptococcus luteciae, certain species of Streptococcus, and other species of Streptococcus.Other), certain species of *Sutterella* spp., certain species of *Turicibacter* spp., *UC Bulleidia*, *UC Enterobacteriaceae*, *UC Faecalibacterium*, *UC Parabacteroides*, *UC Pediococcus*, certain species of *Varibaculum* spp., certain species of *Veillonella* spp., *Sutterella*, *Turicibacter*, *UC Clostridiales*, *UC Erysipelotrichaceae*, *UC Ruminococcaceae*, *Veillonella parvula*, certain species of *Veillonella* *Veillonella dispar* and *Weissella*. In some implementations, polynucleotides are integrated into the genome of the bacterial cell.

[0018] Probiotics

[0019] In one aspect of this disclosure, probiotics are provided. In some embodiments, the probiotics comprise engineered bacterial cells comprising a polynucleotide encoding a sequence encoding a modified *Kristensenia pyrrhizina* bile salt hydrolase (BSH) enzyme, said modified *Kristensenia pyrrhizina* bile salt hydrolase (BSH) enzyme being at least 90% identical to SEQ ID NO: 1 and containing one or more amino acid substitutions at positions A140, N274, C2, F68, L137, H212, I59, S103, K92, K312, L20, V22, L67, P225, or Y26 relative to SEQ ID NO: 1. In some embodiments, the modified enzyme is at least 90% identical to SEQ ID NO: 1 and contains one or more amino acid substitutions at positions C2, Y8, Y34, N46, H47, Y56, I57, Y64, P81, K90, F99, L127, S136, Y176, K189, S206, R207, A221, N292, or R323 relative to SEQ ID NO: 1. In some implementations, one or more alternatives include Y8K, Y8T, Y8Q, Y8V, Y8T, Y34N, Y34S, N46K, H47S, H47N, Y56A, Y56M, Y56T, Y56L, Y56V, I57V, Y64S, Y64F, P81A, K90E, F99Y, L127I, L127V, S136A, S136T, Y176K, K189P, K189N, S206G, R207Q, A221P, A221L, N292S, N292R, N292H, R323P, R323T, R323E, R323L, or R323Q. In some embodiments, one or more substitutions include Y8V, Y34K, I57V, Y64S, P81A, K90E, L127I, S136A, K189P, K189N, S206G, R207Q, N292S, R323L, or R323E. In some embodiments, one or more substitutions include I57V. In some embodiments, one or more substitutions include the modified enzyme according to claim 1, wherein one or more substitutions include Y64S. In some embodiments, one or more substitutions include S136A. In some embodiments, one or more substitutions include K189P. In some embodiments, one or more substitutions include Y8V, P81A, S206G, and R207Q. In some embodiments, one or more substitutions include Y34K, I57V, L127I, and N292S. In some embodiments, one or more substitutions include Y56T, I57V, L127I, and N292S. In some implementations, one or more replacements include K90E, L127I,N292S and R323E. In some embodiments, one or more substitutions include Y34N, N46K, H47N, Y64F, Y176K, and A221L. In some embodiments, one or more substitutions include Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L. In some embodiments, one or more substitutions include C2S. In some embodiments, the modified enzyme comprises or consists of one of SEQ ID NO: 2-47. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 5, and wherein the modified enzyme has the substitution I57V relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 5. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 6, and wherein the modified enzyme has a substituted Y64S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 6. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 8, and wherein the modified enzyme has a substituted S136A relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 9, and wherein the modified enzyme has a substituted K189P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 36, and wherein the modified enzyme has substitutions of Y8V, P81A, S206G, and R207Q relative to SEQ ID NO: 1. Optionally, the modified enzyme comprises or is composed of SEQ ID NO: 36. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 37, and wherein the modified enzyme has substitutions of Y34K, I57V, and R207Q relative to SEQ ID NO: 1.L127I and N292S, optionally, wherein the modified enzyme comprises or is composed of SEQ ID NO: 37. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 41, and wherein the modified enzyme has substitutions of Y56T, I57V, L127I, and N292S relative to SEQ ID NO: 1, optionally, wherein the modified enzyme comprises or is composed of SEQ ID NO: 41. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 42, and wherein the modified enzyme has substitutions of K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1, optionally, wherein the modified enzyme comprises or is composed of SEQ ID NO: 42. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 43, and wherein the modified enzyme has substitutions of Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 43. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 44, and wherein the modified enzyme has substitutions of Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 44. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 45, and wherein the modified enzyme has substitutions of Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N relative to SEQ ID NO:1.N292S and R323L, optionally, wherein the modified enzyme comprises or is composed of SEQ ID NO: 45. In some embodiments, the modified enzyme has increased activity compared to wild-type *Kristensenia spp.* BSH at about pH 5. In some embodiments, the modified enzyme has increased activity compared to wild-type *Kristensenia spp.* BSH at about pH 7. In some embodiments, the modified *Kristensenia spp.* bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, wherein the modified BSH enzyme has at least one substitution modification relative to SEQ ID NO: 1. In some embodiments, the polynucleotide further comprises one or more regulatory elements, wherein the regulatory elements are operatively linked to the sequence encoding the modified enzyme. In some embodiments, the one or more regulatory elements comprise a promoter and / or an enhancer. In some embodiments, the polynucleotide further comprises a selection marker. In some embodiments, the one or more regulatory elements are promoters, and the promoters are constitutive promoters. In some embodiments, one or more regulatory elements are promoters, and the promoters are inducible promoters. In some embodiments, the promoters may be induced by environmental conditions or in response to hypoxic or anaerobic conditions. In some embodiments, the polynucleotide comprises one of SEQ ID NO: 48-79. In some embodiments, the expression of the polynucleotide is operatively linked to a foreign promoter not found in the native Kristensenia pygmaea genome. In some implementations, the engineered bacterial cells are selected from the group consisting of: certain species of *Aminococcus*, certain species of *Actinomyces*, *Ackermania*, certain species of *Isobacterium*, certain species of *Anaerobic Coccus*, certain species of *Anaerobic Bacillus*, certain species of *Bacteroides*, other species of *Bacteroides*, *Bacteroides acidogenic*, *Bacteroides foetida*, *Bacteroides fragilis*, *Bacteroides ovatus*, *Bacteroides monomorpha*, certain species of *Pasteurella*, *Bifidobacterium adolescentis*, other species of *Bifidobacterium*, certain species of *Bifidobacterium*, certain species of *Biliobacterium*, *Brutella ovatus*, *Brutella brevicula*, other species of *Brutella*, certain species of *Brutella*, certain species of *Brutella*, certain species of *Streptococcus*, and certain species of *Christsenia*. Species, certain species of *Citrobacter*, certain species of Clostridium, other species of Clostridium, certain species of Clostridium, *Clostridium perfringens*, certain species of *Clostridium*, other species of *Clostridium*, *Colinella aerogenes*, certain species of *Colinella*, *Colinella faecium*, *Clostridium davidii*, certain species of *Colinella ...Haemophilus parainfluenzae, other species of Haemophilus, certain species of Helicobacter, other species of Helicobacter of the family Trichophytonceae, certain species of Trichophytonceae, Lactobacillus reuteri, Lactobacillus mucosa, Lactobacillus zeatus, certain species of Lactobacillus, certain species of Lactobacillus, certain species of Lactococcus, certain species of Leuconostocaceae, certain species of Megamonas, certain species of Macrococcus, certain species of Methanobacterium, Pseudomonas aeruginosa, certain species of Pseudomonas, Sylvaticus spp., and others. Certain species of *Bacillus*, certain species of *Oscillatoria*, *Pseudomonas difficile*, certain species of *Pseudomonas*, certain species of *Parprevotella*, certain species of *Parprevotella* family, certain species of *Micromonas*, certain species of *Pediococcus*, other species of *Pediococcus*, certain species of *Peptococcus*, certain species of *Peptostreptococcus*, anaerobic *Peptostreptococcus*, other species of *Peptostreptococcus*, certain species of *Koala*, *Prevotella*, certain species of *Prevotella*, *Stecocephala*, Prevostidae, certain species of Proteus, certain species of Rikenaceae, *Roseidonella faecalis*, certain species of *Roseidonella*, other species of Ruminococci, certain species of Ruminococci, *Brugococcus brucelli*, *Active Ruminococcus*, certain species of *Ruminococcus*, other species of *Ruminococcus*, *Chaetomium tortiformis*, certain species of *Slackella*, certain species of S24-7, certain species of SMB53, *Streptococcus pharyngis*, *Streptococcus luteus*, certain species of *Streptococcus*. Other species of *Streptococcus*, certain species of *Sartreus*, certain species of *Bacillus* zurichii, *Bleederella* UC, *Enterobacteraceae* UC, *Femobacterium* UC, *Parabacterium* UC, *Pediococcus* UC, certain species of *Curvibacterium* UC, certain species of *Veillonella* UC, *Sartreus* UC, *Bacillus* UC, *Clostridium* UC, *Erysipelothrix* UC, *Ruminococci* UC, *Veillonella* UC, certain species of *Veillonella* UC, *Veillonella* var. *species*, and *Weissella* UC. In some embodiments, polynucleotides are integrated into the bacterial cell's genome.

[0020] Pharmaceutical compositions containing engineered bacterial cells

[0021] In one aspect of this disclosure, further pharmaceutical compositions are provided. In some embodiments, the pharmaceutical composition comprises engineered bacterial cells comprising a polynucleotide encoding a sequence encoding a modified *Kristensenia pyrrhizina* bile salt hydrolase (BSH) enzyme, said modified *Kristensenia pyrrhizina* bile salt hydrolase (BSH) enzyme being at least 90% identical to SEQ ID NO: 1, and containing one or more amino acid substitutions at positions C2, Y8, Y34, N46, H47, Y56, I57, Y64, P81, K90, F99, L127, S136, Y176, K189, S206, R207, A221, N292, or R323 relative to SEQ ID NO: 1. In some embodiments, the modified enzyme is at least 90% identical to SEQ ID NO: 1, and contains one or more amino acid substitutions at positions C2, Y8, Y34, N46, H47, Y56, I57, Y64, P81, K90, F99, L127, S136, Y176, K189, S206, R207, A221, N292, or R323 relative to SEQ ID NO: 1. In some implementations, one or more alternatives include Y8K, Y8T, Y8Q, Y8V, Y8T, Y34N, Y34S, N46K, H47S, H47N, Y56A, Y56M, Y56T, Y56L, Y56V, I57V, Y64S, Y64F, P81A, K90E, F99Y, L127I, L127V, S136A, S136T, Y176K, K189P, K189N, S206G, R207Q, A221P, A221L, N292S, N292R, N292H, R323P, R323T, R323E, R323L, or R323Q. In some embodiments, one or more substitutions include Y8V, Y34K, I57V, Y64S, P81A, K90E, L127I, S136A, K189P, K189N, S206G, R207Q, N292S, R323L, or R323E. In some embodiments, one or more substitutions include I57V. In some embodiments, one or more substitutions include the modified enzyme according to claim 1, wherein one or more substitutions include Y64S. In some embodiments, one or more substitutions include S136A. In some embodiments, one or more substitutions include K189P. In some embodiments, one or more substitutions include Y8V, P81A, S206G, and R207Q. In some embodiments, one or more substitutions include Y34K, I57V, L127I, and N292S. In some embodiments, one or more substitutions include Y56T, I57V,L127I and N292S. In some embodiments, one or more substitutions include K90E, L127I, N292S, and R323E. In some embodiments, one or more substitutions include Y34N, N46K, H47N, Y64F, Y176K, and A221L. In some embodiments, one or more substitutions include Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L. In some embodiments, one or more substitutions include C2S. In some embodiments, the modified enzyme comprises or consists of one of SEQ ID NO: 2-47. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 5, and wherein the modified enzyme has a substitution I57V relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 5. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 6, and wherein the modified enzyme has a substitution Y64S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 6. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 8, and wherein the modified enzyme has a substitution S136A relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 9, and wherein the modified enzyme has a substituted K189P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 9. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 36, and wherein the modified enzyme has substituted Y8V, P81A, S206G, and R207Q relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 36. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 37, and wherein the modified enzyme has substituted Y34K, I57V, ...L127I and N292S, optionally, wherein the modified enzyme comprises or is composed of SEQ ID NO: 37. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 41, and wherein the modified enzyme has substitutions of Y56T, I57V, L127I, and N292S relative to SEQ ID NO: 1, optionally, wherein the modified enzyme comprises or is composed of SEQ ID NO: 41. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 42, and wherein the modified enzyme has substitutions of K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1, optionally, wherein the modified enzyme comprises or is composed of SEQ ID NO: 42. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 43, and wherein the modified enzyme has substitutions of Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 43. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 44, and wherein the modified enzyme has substitutions of Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 44. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 45, and wherein the modified enzyme has substitutions of Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N relative to SEQ ID NO: 1.N292S and R323L, optionally, wherein the modified enzyme comprises or is composed of SEQ ID NO: 45. In some embodiments, the modified enzyme has increased activity compared to wild-type *Kristensenia spp.* BSH at about pH 5. In some embodiments, the modified enzyme has increased activity compared to wild-type *Kristensenia spp.* BSH at about pH 7. In some embodiments, the modified *Kristensenia spp.* bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, wherein the modified BSH enzyme has at least one substitution modification relative to SEQ ID NO: 1. In some embodiments, the polynucleotide further comprises one or more regulatory elements, wherein the regulatory elements are operatively linked to the sequence encoding the modified enzyme. In some embodiments, the one or more regulatory elements comprise a promoter and / or an enhancer. In some embodiments, the polynucleotide further comprises a selection marker. In some embodiments, the one or more regulatory elements are promoters, and the promoters are constitutive promoters. In some embodiments, one or more regulatory elements are promoters, and the promoters are inducible promoters. In some embodiments, the promoters may be induced by environmental conditions or in response to hypoxic or anaerobic conditions. In some embodiments, the polynucleotide comprises one of SEQ ID NO: 48-79. In some embodiments, the expression of the polynucleotide is operatively linked to a foreign promoter not found in the native Kristensenia pygmaea genome. In some implementations, the engineered bacterial cells are selected from the group consisting of: certain species of *Aminococcus*, certain species of *Actinomyces*, *Ackermania*, certain species of *Isobacterium*, certain species of *Anaerobic Coccus*, certain species of *Anaerobic Bacillus*, certain species of *Bacteroides*, other species of *Bacteroides*, *Bacteroides acidogenic*, *Bacteroides foetida*, *Bacteroides fragilis*, *Bacteroides ovatus*, *Bacteroides monomorpha*, certain species of *Pasteurella*, *Bifidobacterium adolescentis*, other species of *Bifidobacterium*, certain species of *Bifidobacterium*, certain species of *Biliobacterium*, *Brutella ovatus*, *Brutella brevicula*, other species of *Brutella*, certain species of *Brutella*, certain species of *Brutella*, certain species of *Streptococcus*, certain species of *Christsenia*, and *Citrus*. Certain species of *Clostridium*, certain species of Clostridium, other species of Clostridium, certain species of Clostridium, *Clostridium perfringens*, certain species of *Clostridium*, other species of *Clostridium*, *Colinella aerogenes*, certain species of *Colinella*, *Colinella faecalis*, *Clostridium davidii*, certain species of *Colinella*, *Colinella davidii*, certain species of *Colinella ... *Colinella parainfluenzae*, certain species of *Colinella*, certain species of *Colinella*, certain species of *Colinella*, certain species of *Colinella*, certain species of *Colinella*, *Colinella parainfluenzae*, certain species of *Colinella*, certain species of *Colinella*, certain species of *Colinella*, certain species of *Colinella*, *Colinella parainfluenzae*, certain species of *Colinella*,Other species of Haemophilus, certain species of Helicobacter, other species of Helicobacter of the family Trichophytonceae, certain species of Trichophytonceae, Lactobacillus reuteri, Lactobacillus mucosa, Lactobacillus zeatus, certain species of Lactobacillus, certain species of Lactobacillus, certain species of Lactococcus, certain species of Leuconostocaceae, certain species of Megamonas, certain species of Macrococcus, certain species of Methanobacterium, Pyroclasticum, certain species of Pyroclasticum, Sylvaticum spp., certain species of Sylvaticum spp. Species, certain species of *Vibrio*, *Pseudomonas difficile*, certain species of *Pseudomonas*, certain species of *Pleurotus*, certain species of *Pleurotus*aceae, certain species of *Micromonobacter*, certain species of *Pediococcus*, other species of *Pediococcus*, certain species of *Peptococcus*, certain species of *Peptostreptococcus*, anaerobic *Peptostreptococcus*, other species of *Peptostreptococcus*, certain species of *Koala*, *Prevotella*, certain species of *Prevotella*, *Styloprecor*, *Prevotella* Family: Bacteriaceae, certain species of Proteus, certain species of Riken Bacteriaceae, *Roseidonella faecalis*, certain species of *Roseidonella*, other species of Ruminococci, certain species of Ruminococci, *Brugococcus brucelli*, *Active Ruminococcus*, certain species of *Ruminococcus*, other species of *Ruminococcus*, *Chaetomium tortiformis*, certain species of *Slackella*, certain species of S24-7, certain species of SMB53, *Streptococcus pharyngis*, *Streptococcus luteus*, certain species of *Streptococcus*, *Streptococcus* Other species of the genus *Cocci*, certain species of the genus *Sartreus*, certain species of the genus *Zurichobacterium*, UC *Bredella*, UC *Enterobacteriaceae*, UC *Femtobacter*, UC *Parabacteroides*, UC *Pediococcus*, certain species of the genus *Curvibacterium*, certain species of the genus *Veillonella*, *Sartreus*, *Zurichobacterium*, UC *Clostridium*, UC *Erysipelothrix*, UC *Ruminococci*, *Veillonella spp.*, certain species of *Veillonella*, *Veillonella spp.*, and *Weissella*. In some embodiments, polynucleotides are integrated into the genome of the bacterial cell.

[0022] method

[0023] In one aspect of this disclosure, a method is provided. In some embodiments, the method includes administering a pharmaceutical composition comprising a modified BSH enzyme to a subject in need, said modified BSH enzyme being at least 90% identical to SEQ ID NO: 1 and comprising one or more amino acid substitutions at positions C2, Y8, Y34, N46, H47, Y56, I57, Y64, P81, K90, F99, L127, S136, Y176, K189, S206, R207, A221, N292, or R323 relative to SEQ ID NO: 1. In some implementations, one or more alternatives include Y8K, Y8T, Y8Q, Y8V, Y8T, Y34N, Y34S, N46K, H47S, H47N, Y56A, Y56M, Y56T, Y56L, Y56V, I57V, Y64S, Y64F, P81A, K90E, F99Y, L127I, L127V, S136A, S136T, Y176K, K189P, K189N, S206G, R207Q, A221P, A221L, N292S, N292R, N292H, R323P, R323T, R323E, R323L, or R323Q. In some embodiments, one or more substitutions include Y8V, Y34K, I57V, Y64S, P81A, K90E, L127I, S136A, K189P, K189N, S206G, R207Q, N292S, R323L, or R323E. In some embodiments, one or more substitutions include I57V. In some embodiments, one or more substitutions include the modified enzyme according to claim 1, wherein one or more substitutions include Y64S. In some embodiments, one or more substitutions include S136A. In some embodiments, one or more substitutions include K189P. In some embodiments, one or more substitutions include Y8V, P81A, S206G, and R207Q. In some embodiments, one or more substitutions include Y34K, I57V, L127I, and N292S. In some embodiments, one or more substitutions include Y56T, I57V, L127I, and N292S. In some embodiments, one or more substitutions include K90E, L127I, N292S, and R323E. In some embodiments, one or more substitutions include Y34N, N46K, H47N, Y64F, Y176K, and A221L. In some embodiments, one or more substitutions include Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L. In some embodiments, one or more substitutions include C2S.In some embodiments, the modified enzyme comprises or consists of one of SEQ ID NO: 2-47. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 5, and wherein the modified enzyme has a substitution I57V relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 5. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 6, and wherein the modified enzyme has a substitution Y64S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 6. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 8, and wherein the modified enzyme has a substitution S136A relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 9, and wherein the modified enzyme has a substituted K189P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 36, and wherein the modified enzyme has substituted Y8V, P81A, S206G, and R207Q relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 36. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 37, and wherein the modified enzyme has substituted Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 37. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 41, and wherein the modified enzyme has substitutions of Y56T, I57V, L127I and N292S relative to SEQ ID NO: 1, optionally wherein the modified enzyme comprises or is composed of SEQ ID NO: 41.In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 42, and wherein the modified enzyme has substitutions of K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 42. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 43, and wherein the modified enzyme has substitutions of Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 43. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 44, and wherein the modified enzyme has substitutions of Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1. Optionally, the modified enzyme comprises or is composed of SEQ ID NO: 44. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 45, and wherein the modified enzyme has substitutions of Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1. Optionally, the modified enzyme comprises or is composed of SEQ ID NO: 45. In some embodiments, the modified enzyme exhibits increased activity compared to wild-type *Kristensenia pygmaea* BSH at approximately pH 5. In some embodiments, the modified enzyme exhibits increased activity compared to wild-type *Kristensenia pygmaea* BSH at approximately pH 7. In some embodiments, the modified *Kristensenia pygmaea* bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, wherein the modified BSH enzyme has at least one substitution modification relative to SEQ ID NO: 1. In some embodiments, the subject in need suffers from one or more of the following: non-alcoholic fatty liver disease (NAFLD), a disease or condition associated with metabolic syndrome, or cardiovascular disease.

[0024] In some embodiments, a method of treating non-alcoholic fatty liver disease (NAFLD) in a subject in need is provided, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a modified enzyme to the subject for treating NAFLD, said modified enzyme being at least 90% identical to SEQ ID NO: 1 and comprising one or more amino acid substitutions at positions C2, Y8, Y34, N46, H47, Y56, I57, Y64, P81, K90, F99, L127, S136, Y176, K189, S206, R207, A221, N292, or R323 relative to SEQ ID NO: 1. In some implementations, one or more alternatives include Y8K, Y8T, Y8Q, Y8V, Y8T, Y34N, Y34S, N46K, H47S, H47N, Y56A, Y56M, Y56T, Y56L, Y56V, I57V, Y64S, Y64F, P81A, K90E, F99Y, L127I, L127V, S136A, S136T, Y176K, K189P, K189N, S206G, R207Q, A221P, A221L, N292S, N292R, N292H, R323P, R323T, R323E, R323L, or R323Q. In some embodiments, one or more substitutions include Y8V, Y34K, I57V, Y64S, P81A, K90E, L127I, S136A, K189P, K189N, S206G, R207Q, N292S, R323L, or R323E. In some embodiments, one or more substitutions include I57V. In some embodiments, one or more substitutions include the modified enzyme according to claim 1, wherein one or more substitutions include Y64S. In some embodiments, one or more substitutions include S136A. In some embodiments, one or more substitutions include K189P. In some embodiments, one or more substitutions include Y8V, P81A, S206G, and R207Q. In some embodiments, one or more substitutions include Y34K, I57V, L127I, and N292S. In some embodiments, one or more substitutions include Y56T, I57V, L127I, and N292S. In some embodiments, one or more substitutions include K90E, L127I, N292S, and R323E. In some embodiments, one or more substitutions include Y34N, N46K, H47N, Y64F, Y176K, and A221L. In some embodiments, one or more substitutions include Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L. In some embodiments, one or more substitutions include C2S.In some embodiments, the modified enzyme comprises or consists of one of SEQ ID NO: 2-47. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 5, and wherein the modified enzyme has a substitution I57V relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 5. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 6, and wherein the modified enzyme has a substitution Y64S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 6. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 8, and wherein the modified enzyme has a substitution S136A relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 9, and wherein the modified enzyme has a substituted K189P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 36, and wherein the modified enzyme has substituted Y8V, P81A, S206G, and R207Q relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 36. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 37, and wherein the modified enzyme has substituted Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 37. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 41, and wherein the modified enzyme has substitutions of Y56T, I57V, L127I and N292S relative to SEQ ID NO: 1, optionally wherein the modified enzyme comprises or is composed of SEQ ID NO: 41.In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 42, and wherein the modified enzyme has substitutions of K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 42. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 43, and wherein the modified enzyme has substitutions of Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 43. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 44, and wherein the modified enzyme has substitutions of Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 44. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 45, and wherein the modified enzyme has substitutions of Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 45. In some embodiments, the modified enzyme exhibits increased activity compared to wild-type *Kristensenia spp.* BSH at approximately pH 5. In some embodiments, the modified enzyme exhibits increased activity compared to wild-type *Kristensenia spp.* BSH at approximately pH 7. In some embodiments, the modified *Kristensenia spp.* bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, wherein the modified BSH enzyme has at least one substitution modification relative to SEQ ID NO: 1.

[0025] In some embodiments, a method of treating non-alcoholic steatohepatitis (NASH) in a subject in need is provided, and the method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising a modified enzyme to the subject for treating NASH, said modified enzyme being at least 90% identical to SEQ ID NO: 1 and comprising one or more amino acid substitutions relative to SEQ ID NO: 1 at positions C2, Y8, Y34, N46, H47, Y56, I57, Y64, P81, K90, F99, L127, S136, Y176, K189, S206, R207, A221, N292, or R323. In some implementations, one or more alternatives include Y8K, Y8T, Y8Q, Y8V, Y8T, Y34N, Y34S, N46K, H47S, H47N, Y56A, Y56M, Y56T, Y56L, Y56V, I57V, Y64S, Y64F, P81A, K90E, F99Y, L127I, L127V, S136A, S136T, Y176K, K189P, K189N, S206G, R207Q, A221P, A221L, N292S, N292R, N292H, R323P, R323T, R323E, R323L, or R323Q. In some embodiments, one or more substitutions include Y8V, Y34K, I57V, Y64S, P81A, K90E, L127I, S136A, K189P, K189N, S206G, R207Q, N292S, R323L, or R323E. In some embodiments, one or more substitutions include I57V. In some embodiments, one or more substitutions include the modified enzyme according to claim 1, wherein one or more substitutions include Y64S. In some embodiments, one or more substitutions include S136A. In some embodiments, one or more substitutions include K189P. In some embodiments, one or more substitutions include Y8V, P81A, S206G, and R207Q. In some embodiments, one or more substitutions include Y34K, I57V, L127I, and N292S. In some embodiments, one or more substitutions include Y56T, I57V, L127I, and N292S. In some embodiments, one or more substitutions include K90E, L127I, N292S, and R323E. In some embodiments, one or more substitutions include Y34N, N46K, H47N, Y64F, Y176K, and A221L. In some embodiments, one or more substitutions include Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L. In some embodiments, one or more substitutions include C2S.In some embodiments, the modified enzyme comprises or consists of one of SEQ ID NO: 2-47. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 5, and wherein the modified enzyme has a substitution I57V relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 5. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 6, and wherein the modified enzyme has a substitution Y64S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 6. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 8, and wherein the modified enzyme has a substitution S136A relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 9, and wherein the modified enzyme has a substituted K189P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 36, and wherein the modified enzyme has substituted Y8V, P81A, S206G, and R207Q relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 36. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 37, and wherein the modified enzyme has substituted Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 37. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 41, and wherein the modified enzyme has substitutions of Y56T, I57V, L127I and N292S relative to SEQ ID NO: 1, optionally wherein the modified enzyme comprises or is composed of SEQ ID NO: 41.In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 42, and wherein the modified enzyme has substitutions of K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 42. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 43, and wherein the modified enzyme has substitutions of Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 43. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 44, and wherein the modified enzyme has substitutions of Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 44. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 45, and wherein the modified enzyme has substitutions of Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 45. In some embodiments, the modified enzyme exhibits increased activity compared to wild-type *Kristensenia spp.* BSH at approximately pH 5. In some embodiments, the modified enzyme exhibits increased activity compared to wild-type *Kristensenia spp.* BSH at approximately pH 7. In some embodiments, the modified *Kristensenia spp.* bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, wherein the modified BSH enzyme has at least one substitution modification relative to SEQ ID NO: 1.

[0026] In some embodiments, a method of treating liver cancer in a subject in need is provided, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a modified enzyme to the subject for treating liver cancer, said modified enzyme being at least 90% identical to SEQ ID NO: 1 and comprising one or more amino acid substitutions at positions C2, Y8, Y34, N46, H47, Y56, I57, Y64, P81, K90, F99, L127, S136, Y176, K189, S206, R207, A221, N292, or R323 relative to SEQ ID NO: 1. In some implementations, one or more alternatives include Y8K, Y8T, Y8Q, Y8V, Y8T, Y34N, Y34S, N46K, H47S, H47N, Y56A, Y56M, Y56T, Y56L, Y56V, I57V, Y64S, Y64F, P81A, K90E, F99Y, L127I, L127V, S136A, S136T, Y176K, K189P, K189N, S206G, R207Q, A221P, A221L, N292S, N292R, N292H, R323P, R323T, R323E, R323L, or R323Q. In some embodiments, one or more substitutions include Y8V, Y34K, I57V, Y64S, P81A, K90E, L127I, S136A, K189P, K189N, S206G, R207Q, N292S, R323L, or R323E. In some embodiments, one or more substitutions include I57V. In some embodiments, one or more substitutions include the modified enzyme according to claim 1, wherein one or more substitutions include Y64S. In some embodiments, one or more substitutions include S136A. In some embodiments, one or more substitutions include K189P. In some embodiments, one or more substitutions include Y8V, P81A, S206G, and R207Q. In some embodiments, one or more substitutions include Y34K, I57V, L127I, and N292S. In some embodiments, one or more substitutions include Y56T, I57V, L127I, and N292S. In some embodiments, one or more substitutions include K90E, L127I, N292S, and R323E. In some embodiments, one or more substitutions include Y34N, N46K, H47N, Y64F, Y176K, and A221L. In some embodiments, one or more substitutions include Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L. In some embodiments, one or more substitutions include C2S.In some embodiments, the modified enzyme comprises or consists of one of SEQ ID NO: 2-47. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 5, and wherein the modified enzyme has a substitution I57V relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 5. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 6, and wherein the modified enzyme has a substitution Y64S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 6. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 8, and wherein the modified enzyme has a substitution S136A relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 9, and wherein the modified enzyme has a substituted K189P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 36, and wherein the modified enzyme has substituted Y8V, P81A, S206G, and R207Q relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 36. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 37, and wherein the modified enzyme has substituted Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 37. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 41, and wherein the modified enzyme has substitutions of Y56T, I57V, L127I and N292S relative to SEQ ID NO: 1, optionally wherein the modified enzyme comprises or is composed of SEQ ID NO: 41.In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 42, and wherein the modified enzyme has substitutions of K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 42. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 43, and wherein the modified enzyme has substitutions of Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 43. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 44, and wherein the modified enzyme has substitutions of Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 44. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 45, and wherein the modified enzyme has substitutions of Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 45. In some embodiments, the modified enzyme exhibits increased activity compared to wild-type *Kristensenia spp.* BSH at approximately pH 5. In some embodiments, the modified enzyme exhibits increased activity compared to wild-type *Kristensenia spp.* BSH at approximately pH 7. In some embodiments, the modified *Kristensenia spp.* bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, wherein the modified BSH enzyme has at least one substitution modification relative to SEQ ID NO: 1.

[0027] In some embodiments, a method of treating Alzheimer's disease in a subject in need is provided, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a modified enzyme to the subject for treating Alzheimer's disease, said modified enzyme being at least 90% identical to SEQ ID NO: 1 and comprising one or more amino acid substitutions at positions C2, Y8, Y34, N46, H47, Y56, I57, Y64, P81, K90, F99, L127, S136, Y176, K189, S206, R207, A221, N292, or R323 relative to SEQ ID NO: 1. In some implementations, one or more alternatives include Y8K, Y8T, Y8Q, Y8V, Y8T, Y34N, Y34S, N46K, H47S, H47N, Y56A, Y56M, Y56T, Y56L, Y56V, I57V, Y64S, Y64F, P81A, K90E, F99Y, L127I, L127V, S136A, S136T, Y176K, K189P, K189N, S206G, R207Q, A221P, A221L, N292S, N292R, N292H, R323P, R323T, R323E, R323L, or R323Q. In some embodiments, one or more substitutions include Y8V, Y34K, I57V, Y64S, P81A, K90E, L127I, S136A, K189P, K189N, S206G, R207Q, N292S, R323L, or R323E. In some embodiments, one or more substitutions include I57V. In some embodiments, one or more substitutions include the modified enzyme according to claim 1, wherein one or more substitutions include Y64S. In some embodiments, one or more substitutions include S136A. In some embodiments, one or more substitutions include K189P. In some embodiments, one or more substitutions include Y8V, P81A, S206G, and R207Q. In some embodiments, one or more substitutions include Y34K, I57V, L127I, and N292S. In some embodiments, one or more substitutions include Y56T, I57V, L127I, and N292S. In some embodiments, one or more substitutions include K90E, L127I, N292S, and R323E. In some embodiments, one or more substitutions include Y34N, N46K, H47N, Y64F, Y176K, and A221L. In some embodiments, one or more substitutions include Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L. In some embodiments, one or more substitutions include C2S.In some embodiments, the modified enzyme comprises or consists of one of SEQ ID NO: 2-47. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 5, and wherein the modified enzyme has a substitution I57V relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 5. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 6, and wherein the modified enzyme has a substitution Y64S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 6. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 8, and wherein the modified enzyme has a substitution S136A relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 9, and wherein the modified enzyme has a substituted K189P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 36, and wherein the modified enzyme has substituted Y8V, P81A, S206G, and R207Q relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 36. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 37, and wherein the modified enzyme has substituted Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 37. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 41, and wherein the modified enzyme has substitutions of Y56T, I57V, L127I and N292S relative to SEQ ID NO: 1, optionally wherein the modified enzyme comprises or is composed of SEQ ID NO: 41.In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 42, and wherein the modified enzyme has substitutions of K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 42. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 43, and wherein the modified enzyme has substitutions of Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 43. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 44, and wherein the modified enzyme has substitutions of Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 44. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 45, and wherein the modified enzyme has substitutions of Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 45. In some embodiments, the modified enzyme exhibits increased activity compared to wild-type *Kristensenia spp.* BSH at approximately pH 5. In some embodiments, the modified enzyme exhibits increased activity compared to wild-type *Kristensenia spp.* BSH at approximately pH 7. In some embodiments, the modified *Kristensenia spp.* bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, wherein the modified BSH enzyme has at least one substitution modification relative to SEQ ID NO: 1.

[0028] In some embodiments, a method is provided for treating a disease or condition associated with metabolic syndrome in a subject in need, and the method includes administering a therapeutically effective amount of a pharmaceutical composition comprising a modified enzyme to the subject for treating a disease or condition associated with metabolic syndrome, said modified enzyme being at least 90% identical to SEQ ID NO: 1 and comprising one or more amino acid substitutions at positions C2, Y8, Y34, N46, H47, Y56, I57, Y64, P81, K90, F99, L127, S136, Y176, K189, S206, R207, A221, N292, or R323 relative to SEQ ID NO: 1. In some implementations, one or more alternatives include Y8K, Y8T, Y8Q, Y8V, Y8T, Y34N, Y34S, N46K, H47S, H47N, Y56A, Y56M, Y56T, Y56L, Y56V, I57V, Y64S, Y64F, P81A, K90E, F99Y, L127I, L127V, S136A, S136T, Y176K, K189P, K189N, S206G, R207Q, A221P, A221L, N292S, N292R, N292H, R323P, R323T, R323E, R323L, or R323Q. In some embodiments, one or more substitutions include Y8V, Y34K, I57V, Y64S, P81A, K90E, L127I, S136A, K189P, K189N, S206G, R207Q, N292S, R323L, or R323E. In some embodiments, one or more substitutions include I57V. In some embodiments, one or more substitutions include the modified enzyme according to claim 1, wherein one or more substitutions include Y64S. In some embodiments, one or more substitutions include S136A. In some embodiments, one or more substitutions include K189P. In some embodiments, one or more substitutions include Y8V, P81A, S206G, and R207Q. In some embodiments, one or more substitutions include Y34K, I57V, L127I, and N292S. In some embodiments, one or more substitutions include Y56T, I57V, L127I, and N292S. In some embodiments, one or more substitutions include K90E, L127I, N292S, and R323E. In some embodiments, one or more substitutions include Y34N, N46K, H47N, Y64F, Y176K, and A221L. In some embodiments, one or more substitutions include Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L. In some embodiments, one or more substitutions include C2S.In some embodiments, the modified enzyme comprises or consists of one of SEQ ID NO: 2-47. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 5, and wherein the modified enzyme has a substitution I57V relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 5. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 6, and wherein the modified enzyme has a substitution Y64S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 6. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 8, and wherein the modified enzyme has a substitution S136A relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 9, and wherein the modified enzyme has a substituted K189P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 36, and wherein the modified enzyme has substituted Y8V, P81A, S206G, and R207Q relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 36. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 37, and wherein the modified enzyme has substituted Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or consists of SEQ ID NO: 37. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 41, and wherein the modified enzyme has substitutions of Y56T, I57V, L127I and N292S relative to SEQ ID NO: 1, optionally wherein the modified enzyme comprises or is composed of SEQ ID NO: 41.In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 42, and wherein the modified enzyme has substitutions of K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 42. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 43, and wherein the modified enzyme has substitutions of Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 43. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 44, and wherein the modified enzyme has substitutions of Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 44. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 45, and wherein the modified enzyme has substitutions of Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1; optionally, the modified enzyme comprises or is composed of SEQ ID NO: 45. In some embodiments, the modified enzyme exhibits increased activity compared to wild-type *Kristensenia spp.* BSH at approximately pH 5. In some embodiments, the modified enzyme exhibits increased activity compared to wild-type *Kristensenia spp.* BSH at approximately pH 7. In some embodiments, the modified *Kristensenia spp.* bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, wherein the modified BSH enzyme has at least one substitution modification relative to SEQ ID NO: 1. In some embodiments, the disease or condition associated with metabolic syndrome is selected from the group consisting of: hyperlipidemia, hypercholesterolemia, obesity, and cardiovascular disease. In some embodiments, the cardiovascular disease is selected from the group consisting of: coronary artery disease, peripheral artery disease, carotid artery disease, heart failure, and stroke.

[0029] In one aspect of this disclosure, a medical food is provided. In some embodiments, the medical food comprises the modified BSH enzyme of this disclosure, the prebiotic of this disclosure, the probiotic of this disclosure, or the bacterial cells of this disclosure.

[0030] In one aspect of this disclosure, a method for preparing a modified bile salt hydrolase (BSH) enzyme is provided, and the method includes introducing the polynucleotides of this disclosure into cells, thereby causing the cells to express the modified BSH enzyme. In some embodiments, the method further includes enriching, purifying, or isolating the modified BSH enzyme. In some embodiments, the cells are mammalian cells, insect cells, fungal cells, or bacterial cells. Attached Figure Description

[0031] Figure 1 An exemplary enzymatic reaction with the disclosed BSH enzyme is shown.

[0032] Figure 2 An exemplary enzymatic reaction with the disclosed BSH enzyme is shown.

[0033] Figure 3A and 3B Exemplary data demonstrating GCA hydrolysis are shown using the disclosed enzyme and glycine assays.

[0034] Figure 4A and 4B Exemplary data demonstrating the hydrolysis of GCDCA by assays of the disclosed enzymes and glycine are shown.

[0035] Figure 5A , 5B Images 5C, 5D, 5E, and 5F show exemplary polyacrylamide gels demonstrating successful expression of the indicated modified BSH enzyme. Staining: Fixation was performed for 60 minutes in staining solution (25% (v / v) isopropanol, 10% (v / v) acetic acid, 65% (v / v) H2O), followed by staining for 60 minutes in staining solution (10% acetic acid (v / v), 0.006% (w / v) Coomassie Brilliant Blue R-250, 90% H2O). 1_BSH_WT is an irrelevant BSH enzyme control different from the BSH of *Kristensenia minora* disclosed herein. BSH2_WT is WT *Kristensenia minora* BSH, i.e., SEQ ID NO: 1.

[0036] Figure 6A and 6B A pie chart depicting the balance of BA in healthy, NASH, and NAFLD states is shown. The data in the chart is from Puri, P. et al., THE PRESENCE AND SEVERITY OF NONALCOHOLIC STEATOHEPATITIS ISASSOCIATED WITH SPECIFIC CHANGES IN CIRCULATING BILE ACIDS. Hepatology. 2018 Feb; 67(2): 534–548, which is incorporated herein by reference in its entirety.

[0037] Figure 7 The pie chart illustrates the novel method disclosed herein for reversing BA balance from a disease state back to a healthy state using a novel enzyme with increased BA debinding activity. Detailed Implementation

[0038] This disclosure provides a modified *Kristensenium micranthum* bile salt hydrolase (BSH) enzyme, a pharmaceutical composition comprising the modified BSH enzyme, a polynucleotide encoding the modified BSH enzyme, a cell (including but not limited to bacterial cells) comprising the modified BSH enzyme, a cell (including but not limited to bacterial cells) comprising the disclosed polynucleotide, and methods for using the modified BSH enzyme, cells (e.g., bacterial cells), and pharmaceutical composition as a probiotic or therapeutic agent for treating human and animal diseases, improving the health and growth of livestock and companion animals, agriculture, and biofuel applications. The disclosed compositions and methods aim to shift bile acid balance to a healthy state while maintaining the integrity of the bile acid pool.

[0039] Bile salt hydrolases catalyze the hydrolysis of "bound" bile acids into "unbound" bile acids, releasing amino acids such as glycine or taurine. For example, it has been experimentally demonstrated that the disclosed modified BSH enzymes hydrolyze the bile acid glycine-chenodeoxycholic acid (GCDCA) to produce chenodeoxycholic acid and free glycine, and hydrolyze glycine-chenodeoxycholic acid to produce cholic acid and glycine. However, it is believed that the disclosed modified BSH enzymes hydrolyze additional types of bile acids.

[0040] As used herein, “engineered BSH” or “modified BSH” are used interchangeably. Furthermore, in the context of this disclosure, unless otherwise stated, BSH is understood to refer to *Christsenium milis* BSH.

[0041] Modified Christensenella pyrenoidosa bile salt hydrolase

[0042] In one aspect of this disclosure, a modified *Christsenium spp.* bile salt hydrolase is provided. The modified enzyme contains one or more amino acid substitutions at positions C2, Y8, Y34, N46, H47, Y56, I57, Y64, P81, K90, F99, L127, S136, Y176, K189, S206, R207, A221, N292, or R323 relative to SEQ ID NO: 1.

[0043] SEQ ID NO: 1 is:

[0044] MCTAITYYTKDHYFGRNLDLEFSYNETVTVTPKYYPFHFRNGKVLNHHYAMIGMAYIVDDFPLYYDATNEKGLSMAGLNFPDNADYKEVKEGYDNIAPFEFIPWILGQCASVSEARILLEQINLVNLNFSEELPLSPLHWMISDQRDSIVVESTKDGLKVFE NPVGVLTNNPTFDYQMFNLNNYMHLSKEPPANTFAAELELEQYSRGMGAIGLPGDLSSASRFVKAAFTKMNSVSGDSESESISQFFHILGSVEQQRGCVHLGEDKYEITIYSSCCNMDKGIYYYTTYENNQITAVDMYKENLDGNTIISYPLMKEQQINYRNY

[0045] As used herein, “substitution” or “substitution modification” is used interchangeably and refers to the substitution of one amino acid for another. The disclosed modified enzyme is at least about 90% identical to SEQ ID NO: 1. The disclosed modified BSH enzyme is 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%, and at least about 99% identical to SEQ ID NO: 1, which is the amino acid sequence of wild-type Christensenella pyrenoidosa bile salt hydrolase. One or more substitutions may include C2S, Y8K, Y8T, Y8Q, Y8V, Y8T, Y34N, Y34S, N46K, H47S, H47N, Y56A, Y56M, Y56T, Y56L, Y56V, I57V, Y64S, Y64F, P81A, K90E, F99Y, L127I, L127V, S136A, S136T, Y176K, K189P, K189N, S206G, R207Q, A221P, A221L, N292S, N292R, N292H, R323P, R323T, R323E, R323L, or R323Q.

[0046] One or more substitutions may include Y8V, Y34K, I57V, Y64S, P81A, K90E, L127I, S136A, K189P, K189N, S206G, R207Q, N292S, R323L, or R323E. One or more substitutions may include I57V. One or more substitutions may include Y64S. One or more substitutions may include S136A. One or more substitutions may include K189P. One or more substitutions may include Y8V, P81A, S206G, and R207Q. One or more substitutions may include Y34K, I57V, L127I, and N292S. One or more substitutions may include Y56T, I57V, L127I, and N292S. One or more substitutions may include K90E, L127I, N292S, and R323E. One or more substitutions may include Y34N, N46K, H47N, Y64F, Y176K, and A221L. One or more substitutions may include Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L. One or more substitutions may include C2S. The modified enzyme may contain one of SEQ ID NO: 2-47. The modified enzyme may contain one of SEQ ID NO: 5, 6, 8, 9, 36, 37, and 41-47.

[0047] The modified enzyme may contain a sequence having 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%, or at least about 99% identity with SEQ ID NO: 5, and wherein the modified enzyme has a substitution I57V relative to SEQ ID NO: 1.

[0048] The modified enzyme may contain a sequence having 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%, or at least about 99% identity with SEQ ID NO: 6, and wherein the modified enzyme has a substituted Y64S relative to SEQ ID NO: 1.

[0049] The modified enzyme may contain a sequence having 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%, or at least about 99% identity with SEQ ID NO: 8, and wherein the modified enzyme has a substitution S136A relative to SEQ ID NO: 1.

[0050] The modified enzyme may contain a sequence having 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%, or at least about 99% identity with SEQ ID NO: 9, and wherein the modified enzyme has a substituted K189P relative to SEQ ID NO: 1.

[0051] The modified enzyme may contain a sequence having 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%, or at least about 99% identity with SEQ ID NO: 36, and wherein the modified enzyme has substitutions of Y8V, P81A, S206G, and R207Q relative to SEQ ID NO: 1.

[0052] The modified enzyme may contain a sequence having 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%, or at least about 99% identity with SEQ ID NO: 37, and wherein the modified enzyme has substitutions of Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1.

[0053] The modified enzyme may contain a sequence having 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%, or at least about 99% identity with SEQ ID NO: 41, and wherein the modified enzyme has substitutions of Y56T, I57V, L127I, and N292S relative to SEQ ID NO: 1.

[0054] The modified enzyme may contain a sequence having 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%, or at least about 99% identity with SEQ ID NO: 42, and wherein the modified enzyme has substitutions of K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1.

[0055] The modified enzyme may contain a sequence having 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%, or at least about 99% identity with SEQ ID NO: 43, and wherein the modified enzyme has substitutions of Y34N, H47N, Y64F, Y176K, or A221P relative to SEQ ID NO: 1.

[0056] The modified enzyme may contain a sequence having 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%, or at least about 99% identity with SEQ ID NO: 1, and wherein the modified enzyme has substitutions of Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1.

[0057] The modified enzyme may comprise a sequence having 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%, and at least about 99% identity with SEQ ID NO: 45, and wherein the modified enzyme has substitutions of Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1.

[0058] The modified enzyme may comprise a sequence having 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%, or at least about 99% identity with any one of SEQ ID NO: 2-47, and wherein the modified enzyme has the substitutions shown in Table 1 relative to SEQ ID NO: 1.

[0059] At approximately pH 5, the modified enzyme may exhibit increased activity compared to wild-type Kristensenium spp. BSH. At approximately pH 7, the modified enzyme may exhibit increased activity compared to wild-type Kristensenium spp. BSH. At approximately pH 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, the modified enzyme may exhibit increased activity compared to wild-type Kristensenium spp. BSH.

[0060] In one aspect of this disclosure, a modified BSH enzyme is provided. The modified BSH enzyme may contain amino acid substitutions at the following positions relative to SEQ ID NO: 1: I5V, Y8R, T9S, D11H, H12T, Y13F, R16M, L18Y, L20A, E21S, F22S, Y24E, N25G, T27K, V28I, T29V, V30I, T31V, K33R, Y34K, F37L, H38K, F39L, R40S, K43E, L45I, N46S, H47N, M54I, Y56K, I57V, V58I, D60N, F61Y, Y64F, Y65K, D66M, T68C, S74A. G77A, L78I, N79S, D82G, N83I, D85S, K87G, V89P, K90T, E91P, Y93K, D94E, I96V, F99Y, F101L, W104Y, Q108R, A110G, S113D, R11 6L, I117E, E120K, Q121T, I122L, L124I, L127V, N128D, E131A, L133H, S136K, L138V, H139K, W140Y, Q145K, R146E, D147K, V151L , S153Q, F161Y, V165T, N170G, F174Y, M178L, K189N, E190K, T195K, A197S, A198P, E199S, L200I, E201K, Q204R, Y205I, R207S, A211N, L218Y, S220D, A221L, R223K, V225I, K226R, A228S, T230V, K231R, M232L, S234A, E240Y, S241D, S243Q, I244L, S245M, G2 52D, E255R, Q256N, Q257V, R258P, C260T, E265G, E269L, I270R, I272L, S274Q, S275T, C277I, N278D, K281R, Y284L, T287R, E290N, N292T, Y300N, E302C, N303D, D305N, N307D, T308K, S311E, Y312F, M315V, K316T, Q318P, N321Y, Y322E, R323L, N324K, and Y325K.The modified enzyme may contain SEQ ID NO: 46, or a sequence having 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%, or at least about 99% identity with SEQ ID NO: 46.

[0061] Modified BSH enzymes may contain amino acid substitutions at the following positions relative to SEQ ID NO: 1: I5V, Y8K, T9S, D11S, H12S, Y13W, R16M, L18Y, L20A, E21S, F22S, Y24E, T27E, V28I, T29V, V30I, T31V, K33R, Y34K, F37L, H38K, F39L, R40S, K43T, L45I, N46D, H47E, Y49N, M54I, Y56E, V58R, D60N, L 63I, Y64F, Y65K, D66M, G77A, L78V, N79S, F80L, D82G, N83I, D85S, K87G, V89K, K90T, Y93K, D94E, F99Y, F10 1L, W104Y, Q108R, C109A, A110S, S113E, R116L, I117K, Q121N, I122L, L124I, L127V, N128D, L133R, S136K, L138V, H139R, W140F, S143A, Q145K, R146D, D147K, S148A, S153Q, F161Y, V165T, N170G, T173L, F174Y, M178L, K189N, E190A, T195K, A198P, E199N, L 200I, E201K, Q204R, Y205I, R207A, M209D, A211N, L218K, S220D, A221L, R2 23K, V225I, K226R, A228T, T230V, K231R, M232L, S234A, S239T, S241D, S24 3Q, I244L, S245M, G252D, E255R, Q256D, Q257V, R258P, C260T, H262W, E265G, E269L, I270R, I272L, S274Q, S275V, C276A, C277I, K281T, I283K, Y284L, T287R, E290N, N292S, T295N, A296M, Y300N, E302C, N303D, D305N, G306S, N307D, T308K, Y312F, M315Q, Q318L, Q319D, N321K, Y322E, R323L, and Y325K.The modified enzyme may contain SEQ ID NO: 47, or a sequence having 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%, or at least about 99% identity with SEQ ID NO: 47.

[0062] The modified enzyme may comprise any one of SEQ ID NO: 2-47, or a sequence having 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%, or at least about 99% identity with any one of SEQ ID NO: 2-47. The modified enzyme may comprise any of the disclosed substitution mutations herein, for example, see Table 1.

[0063] Several BSH mutants of *Christsenia minor* have been generated and are listed below to correspond the experimental nomenclature rules to the characteristics of the mutants. For example, BSH2_3 contains the I57V mutation relative to SEQ ID NO: 1.

[0064] Table 1. Experimental nomenclature rules and characteristics of modified BSH enzymes.

[0065]

[0066]

[0067]

[0068] Now for reference Figure 3A , 3B As described in 4A and 4B, the inventors have elucidated that certain substitutions result in different properties of the modified BSH enzymes of this disclosure. For example, compared to wild-type BSH, BSH2_0, BSH2_4, BSH2_6, BSH2_7, BSH2_40, BSH2_42, BSH2_43, BSH2_44, BSH2_45, BSH2_34, and BSH2_35 exhibit increased GCA hydrolytic activity. Compared to wild-type BSH, BSH2_3, BSH2_4, BSH2_6, BSH2_7, BSH2_39, BSH2_40, BSH2_42, BSH2_43, BSH2_44, BSH2_45, BSH2_35, and BSH2_34 exhibit increased GCDCA hydrolysis.

[0069] As those skilled in the art will understand, the various compartments of the human and non-human animal digestive systems have different levels of acidity / alkalinity. For example, the intraluminal pH changes rapidly from the strong acid in the stomach to approximately pH 6 in the duodenum. The pH gradually increases from pH 6 in the small intestine to approximately pH 7.4 in the terminal ileum. The pH decreases to 5.7 in the cecum but then gradually increases again, reaching pH 6.7 in the rectum. Therefore, the inventors anticipate that the disclosed modified BSH enzymes can have different or complementary applications due to the potential for different pH-related activities.

[0070] The published data also indicate that certain substitutions lead to a decrease or absence of activity in modified BSH enzymes; for example, C2S substitution eliminates enzyme activity. See, for example, Figure 3A , 3B 4A and 4B.

[0071] The disclosed modified BSH enzymes can be produced in recombinant systems, for example, the modified BSH enzymes can be expressed by cells including but not limited to mammalian cells (e.g., human embryonic kidney (HEK) cells), insect cells, fungal cells (e.g., yeast), and bacterial cells (e.g., Escherichia coli).

[0072] By way of example and not limitation, in some embodiments, the modified BSH enzyme of this disclosure may include one or more of the following additional modifications: codon optimization for better expression in a particular system; one or more post-translational modifications; one or more non-natural amino acids; one or more tags or detectable markers; one or more chemical motifs for linking or encapsulating with a solid or semi-solid support (such as beads, carrier molecules, or target delivery molecules (such as proteins or viruses)).

[0073] Polynucleotides

[0074] In one aspect of this disclosure, a polynucleotide is provided. In some embodiments, the polynucleotide comprises a sequence encoding the modified Christensenella pyrenoidosa bile salt hydrolase (BSH) enzyme of this disclosure.

[0075] The polynucleotide may contain one of SEQ ID NO: 48-79. The polynucleotide may contain one or more regulatory elements. As used herein, a "regulatory element" refers to a polynucleotide sequence that regulates the expression of another polynucleotide sequence (e.g., a promoter or enhancer).

[0076] As used herein, "operationally linked" refers to a functional link between two or more sequences such that activity at or on one sequence affects activity at or on other sequences. For example, an operable link between a polynucleotide of interest (e.g., a sequence encoding a modified BSH enzyme disclosed herein) and a regulatory element (e.g., a promoter) is a functional link that allows expression of the polynucleotide of interest.

[0077] The promoter can be a constitutive promoter or an inducible promoter. A wide variety of promoters are known in the art, for example, SEQ ID NO: 48-52 or 58-61. See Table 2 for further details.

[0078] Promoters can be induced by environmental conditions.

[0079] The promoter can respond to low-oxygen or anaerobic conditions.

[0080] Table 2. Exemplary regulating elements and other sequences.

[0081]

[0082] As used herein, “selection marker” refers to any marker that can be used to select the expression of a polynucleotide, such as antibiotic resistance, auxotrophic morphology, fluorescence, etc. Suitable selection markers are known in the art.

[0083] The phrases “% sequence identity,” “percentage identity,” or “% identity” refer to the percentage of amino acid residue matches between at least two amino acid sequences aligned using a normalized algorithm. Methods for amino acid sequence alignment are well known. Some alignment methods consider conserved amino acid substitutions. Such conserved substitutions, explained in more detail below, typically preserve the charge and hydrophobicity at the substitution site, thus preserving the structure (and therefore function) of the polypeptide. Percentage identity of amino acid sequences can be determined as understood in the art. (See, for example, U.S. Patent No. 7,396,664, which is incorporated herein by reference in its entirety). A commonly used and freely available set of sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), which is available from multiple sources, including the NCBI website in Bethesda, Maryland. The BLAST software suite includes various sequence analysis programs, including “blastp,” which is used to align known amino acid sequences with other amino acid sequences from various databases.

[0084] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. These terms refer to proteins, peptides, or polypeptides of any size, structure, or function. Typically, proteins, peptides, or polypeptides will be at least three amino acids long. A protein, peptide, or polypeptide can refer to a single protein or a collection of proteins. One or more amino acids in a protein, peptide, or polypeptide can be non-natural, modified, for example, by adding chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl, isofarnesyl, fatty acid groups, linkers, for conjugation, functionalization, labeling, or other modifications. Proteins, peptides, or polypeptides can also be single molecules or can be multi-molecular complexes. Proteins, peptides, or polypeptides can simply be fragments of naturally occurring proteins or peptides. Proteins, peptides, or polypeptides can be naturally occurring, recombinant, or synthetic, or any combination thereof. Proteins can contain different structural domains, such as nucleic acid-binding domains and nucleic acid-cleaving domains. In some embodiments, a protein contains a proteinaceous part, such as the amino acid sequence constituting the nucleic acid-binding domain.

[0085] The nucleic acids, proteins, and / or other compositions described herein may be purified. As used herein, “purified” means isolated from most other compounds or entities and encompasses partially purified or substantially purified. Purity can be expressed as a weight-to-weight ratio measurement and can be determined using various analytical techniques, such as, but not limited to, mass spectrometry, HPLC, etc.

[0086] Peptide sequence identity can be measured over the entire length of a well-defined peptide sequence, such as as defined by a specific SEQ ID NO, or over a shorter length, such as over the length of a fragment extracted from a larger well-defined peptide sequence, for example, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 consecutive residues. Such lengths are merely exemplary and it should be understood that any fragment length supported by the sequences shown herein in tables, figures, or sequence listings can be used to describe lengths on which percentage identity can be measured.

[0087] The disclosed modified BSH enzymes can be delivered to the subject or host by any of the following methods, including but not limited to: in vivo delivery, static delivery, chemical delivery, nanoparticle delivery, synthetic delivery, etc.

[0088] Method for preparing modified Christensenella pyrenoidosa bile salt hydrolase

[0089] In one aspect of this disclosure, a method for preparing the modified BSH enzyme of this disclosure is provided. In some embodiments, the method for preparing the modified BSH enzyme includes introducing the polynucleotide of this disclosure into cells, wherein the cells express the polynucleotide. The method may further include purifying, enriching, or separating the modified BSH enzyme using methods known in the art, including but not limited to: chromatography, such as liquid chromatography (LC), high-performance liquid chromatography (HPLC), size exclusion chromatography (SEC), affinity chromatography, etc.

[0090] prebiotics

[0091] In one aspect of this disclosure, a prebiotic is provided. In some embodiments, the prebiotic comprises one or more of the disclosed modified BSH enzymes. The prebiotic may further comprise one or more additional ingredients to stabilize or preserve the modified BSH enzymes of this disclosure.

[0092] As used in this article, "prebiotic" refers to an indigestible food component that promotes the growth of beneficial microorganisms in the digestive tract of an animal (e.g., in the intestine).

[0093] It is believed that prebiotics alter the gut microbiome of subjects by increasing the amount of unconjugated bile acids. Furthermore, increasing unconjugated bile acids can improve lipid uptake in livestock, thereby improving livestock growth and health.

[0094] As discussed above, the modified BSH enzymes of this disclosure have different properties and can be selected or combined based on these properties (e.g., activity, selectivity, activity at a specific pH) to be included in prebiotic compositions. The prebiotic may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different types of modified enzymes, i.e., modified BSH enzymes with different substitutions.

[0095] engineered bacterial cells

[0096] In one aspect of this disclosure, engineered bacterial cells are provided. In some embodiments, the bacterial cells comprise a polynucleotide containing a sequence encoding a disclosed modified BSH enzyme. The engineered bacteria are safe, well-tolerated, and can be engrafted in a host to improve the host's microbiome, thereby extending therapeutic benefits.

[0097] The disclosed bacterial cells were engineered to express modified BSH enzymes. It is believed that engineered bacterial cells increased the amount of unconjugated bile acids in subjects, which was associated with a reduced incidence of various human and animal diseases and conditions, including but not limited to non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, metabolic syndrome, and liver cancer.

[0098] Engineered bacterial cells may be, for example, *Christsenium spp.* cells or any other suitable bacterial cells, such as, but not limited to, one or more of the following non-limiting list of exemplary bacterial types that may be used: certain species of *Aminococcus*, certain species of *Actinomyces*, *Ackermania*, certain species of *Isobacterium*, certain species of *Anaerobic Coccus*, certain species of *Anaerobic Bacillus*, certain species of *Bacteroides*, *Bacteroides thetaiotaomicron*, *Bacteroides vulgatus*, *Bacteroides vulgatus* VPI-5482, *Bacteroides fragilis* NCTC-9343, *Bacteroides ovalis*, *Bacteroides monomorpha*, and *Bacteroides escherichia*. Eggerthii), other species of Bacteroides, Bacteroides acid-producing, Bacteroides coccidioides, Bacteroides fragilis, Bacteroides ovatus, Bacteroides monomorphoides, certain species of Pasteurella, Bifidobacterium adolescentis, other species of Bifidobacterium, certain species of Bifidobacterium, certain species of Bileophila, Broutella ovatus, Broutella brevicella, other species of Broutella, certain species of Broutella, certain species of Breedella, certain species of Streptococcus, certain species of Citrobacter, certain species of Clostridium, other species of Clostridium, certain species of Clostridium. Species, Clostridium perfringens, certain species of Clostridium, other species of Clostridium, Collins aerogenes, certain species of Collins, Collins faecalis, Clavicipitolus davidii, certain species of Factococcus, certain species of Rhodotorulaceae, certain species of Desulfovibrioceae, certain species of Alisteria dilatata, Dorperella formateriate, certain species of Dorperella, other species of Dorperella, Egretteella tarda, other species of Enterobacteriaceae, certain species of Enterobacteriaceae, certain species of Enterococcus, certain species of Erysipelothrix, Amyloliquefaciens, Amyloliquefaciens, Amyloliquefaciens, Long Emyloliquefaciens, certain species of Eubacterium, Clostridium plasmidum, Clostridium... Certain species of *Bacillus*, certain species of *Gymnococcus*, *Haemophilus parainfluenzae*, other species of *Haemophilus*, certain species of *Helicobacter*, other species of *Helicobacter* of the *Trichophyton* family, certain species of the *Trichophyton* family, *Lactobacillus reuteri*, *Lactobacillus mucosa*, *Lactobacillus zeatus*, certain species of *Lactobacillus*, certain species of *Lactobacillus*, certain species of *Lactococcus*, certain species of *Leuconostoc* family, certain species of *Methanobacter*, certain species of *Gymnococcus*, certain species of *Gymnococcus*, *Gymnococcus*, *Syntrophus serratifolius*, certain species of *Aureobacter*, certain species of *Vibrio*, *Pseudomonas difficile*, *Pseudomonas parainfluenzae ... Certain species of *Bacteroides*, certain species of *Peptococcus*, certain species of the Paraprevostaceae family, certain species of *Micromonospora*, certain species of *Pediococcus*, other species of *Pediococcus*, certain species of *Peptococcus*, certain species of *Peptostreptococcus*, anaerobic *Peptostreptococcus*, other species of *Peptostreptococcus*, certain species of *Koala*, *Prevostella*, certain species of *Prevostella*, *Stichoprevostella*, Prevostaceae, certain species of *Proteus*, certain species of the Rikenaceae family, *Roseidon*, certain species of *Roseidon*, other species of the Ruminococciaceae family, *Ruminococcus brucelli*,Active rumenococci, certain species of *Ruminococcus*, other species of *Ruminococcus*, *Ruminococcus tortifolius*, certain species of *Slackella*, certain species of S24-7, certain species of SMB53, *Streptococcus pharyngis*, *Streptococcus luteus*, certain species of *Streptococcus*, other species of *Streptococcus*, certain species of *Sartella*, certain species of *Bacillus*, *UC* *Bredella*, *UC* Enterobacteriaceae, *UC* *Femobacter*, *UC* *Parabacter*, *UC* *Pediococcus*, certain species of *Curvibacter*, certain species of *Veillonella*, *Sartella*, *Bacillus*, *UC* Clostridium, *UC* Erysipelothrixceae, *UC* Ruminococciaceae, *Veillonella septembrittleri*, certain species of *Veillonella*, *Veillonella severe*, *s__Turicibacter_sp001543345:1*, and *Weissella*.

[0099] Appropriately, engineered bacterial cells can be selected based on compatibility with a specific host. For example, in the case of engineered bacterial cells designed for administration or delivery to humans, bacteria that are normal components of the human microbiome (e.g., the gut microbiome), such as *Christenia milieus*, can be selected. When *Christenia milieus* is selected as the bacterial cell, the native BSH enzyme in the cell can be disrupted or knocked out, so that only the expression of the modified BSH enzyme is retained.

[0100] Bacterial cells may include polynucleotides containing sequences encoding one or more modified BSH enzymes disclosed herein, said polynucleotides being present and integrated into the bacterial cell genome, or present and ectopically expressed, i.e., not integrated into the bacterial cell genome. Bacterial cells may contain multiple polynucleotides, each polynucleotide containing a nucleic acid sequence encoding one or more modified BSH enzymes disclosed herein.

[0101] The polynucleotide sequence containing the encoding the modified BSH enzyme disclosed herein may contain a "heterologous promoter," that is, a promoter that is not naturally present in a particular bacterial cell. For example, the bacterial cell may be a *Christsenium spp.* cell, and the promoter may be derived from a different organism. In some embodiments, the heterologous promoter is functionally linked to the polynucleotide sequence encoding the modified BSH enzyme, enabling engineered cells to express the modified BSH enzyme.

[0102] Probiotics

[0103] In one aspect of this disclosure, probiotics are provided. In some embodiments, the probiotics comprise engineered bacterial cells of the present disclosure. The probiotics may further comprise one or more carriers or excipients.

[0104] As used herein, “probiotics” refers to live microorganisms intended to provide health benefits to the body when ingested or applied topically. Probiotics can be used in nutritional compositions (which are also disclosed herein). Probiotics may also be referred to as live biotherapeutic products (LBPs).

[0105] For example, the formulation of probiotics to be ingested or delivered is known in the art and is considered routine.

[0106] As discussed above, the disclosed modified enzymes, for example in the form of probiotics containing engineered bacterial cells expressing the modified enzymes, are considered effective in increasing the amount of unconjugated bile acids in the gut of subjects (e.g., human subjects or non-human animal subjects).

[0107] Because the disclosed modified enzymes potentially possess different properties at different pH levels, such as activity and potential specificity, probiotics can comprise more than one type of engineered bacterial cell. For example, probiotics can comprise more than one species of bacterial cells. Probiotics can contain more of a first type of engineered bacterial cells containing a specific modified BSH enzyme and a second type of engineered bacterial cells containing a second, different modified BSH enzyme. Different types of engineered bacterial cells can be selected to occupy different niches within the host's (subject's) microbiome.

[0108] The disclosed probiotics can be used in a variety of methods, for example, the disclosed probiotics can be provided to or administered to a subject.

[0109] The disclosed probiotics can be used in a method to improve livestock productivity, the method comprising providing an effective amount of the disclosed probiotics to improve livestock productivity.

[0110] Livestock can be, for example, ruminants, pigs, birds, fish, crustaceans, or mollusks. Ruminants can be, for example, cows, sheep, goats, deer, buffalo, or camels.

[0111] Animal health

[0112] BSH enzymes in livestock play a crucial role in fat digestion by breaking down bile salts, which is essential for the absorption of dietary fat. This helps improve the animal's nutrient utilization efficiency, as well as its overall health and performance. Although lipid metabolism differs between humans and livestock, BSH remains an important enzyme in gut bacteria. Ruminants such as cattle and sheep have unique digestive systems, including a specialized gastric cavity called the rumen. In the rumen, microbial fermentation of plant material occurs, and the resulting fatty acids and lipids are modified by the gut microbiota before absorption in the lower digestive tract. The modified BSH enzymes of this disclosure, produced by specific gut bacteria in ruminants, can assist in the hydrolysis of bile salts in this context, enabling microorganisms to efficiently metabolize dietary lipids and produce volatile fatty acids that can be absorbed and used as energy. Non-ruminant livestock, such as pigs and poultry, have simpler digestive systems without a rumen. The modified BSH enzymes of this disclosure primarily assist in the digestion of dietary fat in the small intestine in these animals. They hydrolyze bile salts, reducing the effectiveness of bile salts in emulsifying fats, which allows these fats to be efficiently digested and absorbed by the animal. The fatty acid profile can also be affected. In ruminants, the involvement of microbial fermentation and modified BSH enzymes can lead to changes in the fatty acid profile of fats absorbed from the digestive tract. This can result in a higher proportion of volatile fatty acids (e.g., acetate, propionate, butyrate) for the ruminant's own energy and microbial protein synthesis, as well as for milk and meat production.

[0113] In non-ruminant livestock, the administration of modified BSH enzymes can facilitate the efficient absorption of dietary fat, leading to the introduction of a diverse range of fatty acids into the animal's own tissues, which can affect the fat composition of meat and eggs. The role of modified BSH enzymes in livestock can contribute to food security by improving the efficiency, sustainability, and resilience of livestock farming, ultimately leading to increased availability and accessibility of human-edible animal protein sources.

[0114] Modified BSH enzymes can improve the digestion and absorption of dietary fats in livestock. When animals can utilize nutrients in their feed more efficiently, it leads to improved feed conversion efficiency. This means that less feed is needed to produce a given amount of meat, milk, or eggs. Therefore, food resources are used more efficiently, and animal protein production becomes more sustainable.

[0115] Livestock that benefit from improved digestion and health due to the administration of the modified BSH enzymes disclosed herein may exhibit better growth rates, higher milk production, or increased egg production. This increased productivity can lead to greater food output from the same number of animals, thereby contributing to food security by meeting the demand for animal protein.

[0116] Healthy livestock are more resistant to disease, reducing the need for antibiotics and other medications. This, in turn, reduces the risk of antibiotic-resistant pathogens emerging and affecting human health, a key aspect of food safety and security.

[0117] By improving the efficiency of livestock production, the application of the modified BSH enzymes disclosed herein can help reduce the environmental footprint of livestock farming. This can lead to reduced land and resource use, lower greenhouse gas emissions, and less water consumption per unit of food produced. This aligns with sustainable agricultural practices and contributes to long-term food security by ensuring resource availability for future generations.

[0118] Enhanced livestock productivity and health can help reduce food loss in the supply chain. Healthy animals are less likely to suffer from diseases that can lead to waste, and efficient production means less feed is wasted when producing the same amount of meat, milk, or eggs.

[0119] As described in this section, “application” of a modified BSH enzyme may include applying the enzyme as part of a therapeutic / nutritional composition, and / or applying cells expressing the enzyme as part of a therapeutic / nutritional composition.

[0120] Aquaculture

[0121] Although lipid metabolism processes differ significantly (fish have a much simpler digestive system than humans), modern aquaculture techniques still involve feeds containing high levels of fat and lipids. Adding the modified BSH enzymes of this disclosure, for example as prebiotics or probiotics, to their diets can enhance their ability to digest and absorb these nutrients. This can lead to better growth rates and improved feed conversion efficiency, which is important for maximizing the productivity of aquaculture operations. By improving fat and lipid digestibility, the application or provision of the modified BSH enzymes of this disclosure can help fish and other aquatic organisms better utilize the nutrients present in their feed. This can result in more efficient conversion of nutrients into body weight, reduced waste production, and potentially improved water quality in aquaculture systems.

[0122] The modified BSH enzymes disclosed herein allow for the use of optional feed ingredients in aquaculture diets. Some feed ingredients, such as plant proteins and oils, may contain anti-nutritional factors, such as phytates, which bind to minerals and reduce their bioavailability. Providing or administering the modified BSH enzymes of this disclosure can help break down these compounds, enabling the introduction of a wider variety of feed ingredients while maintaining nutritional value.

[0123] Improved nutrient utilization and growth efficiency can lead to reduced environmental impacts of aquaculture operations. If fish and other aquatic organisms grow more efficiently and produce less waste, there will be less nutrient loss and pollution in the surrounding aquatic ecosystem.

[0124] It is believed that providing or administering the modified BSH enzyme of this disclosure can have a positive impact on the health of aquaculture species. Bile salts can possess antimicrobial properties, and by breaking down bile salts, the modified BSH enzyme can potentially reduce the prevalence of harmful pathogens in the gut. This could help reduce the incidence of disease in aquaculture populations.

[0125] Food security

[0126] Providing or administering modified BSH enzymes to livestock can contribute to food security by improving the efficiency, sustainability, and resilience of livestock farming, ultimately leading to increased availability and accessibility of human-edible animal protein sources. Improved nutrient utilization: Modified BSH enzymes can facilitate better digestion and absorption of dietary fats in livestock. When animals can effectively utilize nutrients in their feed, it results in improved feed conversion efficiency. This means that less feed is needed to produce a given amount of meat, milk, or eggs. Therefore, food resources are used more efficiently, and animal protein production becomes more sustainable.

[0127] Livestock benefiting from improved digestion and health due to the application of the modified BSH enzymes disclosed herein may exhibit better growth rates, higher milk production, or increased egg production. This increased productivity can lead to greater grain output from the same number of animals, thus contributing to food security by meeting the demand for animal protein. Enhanced animal health: Healthy livestock are more resistant to disease, reducing the need for antibiotics and other drugs. This, in turn, can reduce the risk of antibiotic-resistant pathogens emerging and affecting human health, a key aspect of food security and food safety.

[0128] By improving the efficiency of livestock production, the application or delivery of the modified BSH enzymes disclosed herein can help reduce the environmental footprint of livestock farming. This can lead to reduced land and resource use, lower greenhouse gas emissions, and less water consumption per unit of food produced. This aligns with sustainable agricultural practices and contributes to long-term food security by ensuring resource availability for future generations. Enhanced livestock productivity and health can help reduce food loss in the supply chain. Healthy animals are less susceptible to diseases that could lead to waste, and efficient production means less feed waste when producing the same amount of meat, milk, or eggs.

[0129] Bioremediation

[0130] The modified BSH enzymes disclosed herein can be used in bioremediation processes to help decompose and remove contaminants from water and soil. Some contaminants, such as certain hydrophobic organic compounds, may be difficult to degrade naturally. The modified BSH enzymes of this disclosure can help enhance the degradation of these compounds by altering their interactions with bile salts and microbial communities.

[0131] Reagent test kit

[0132] In one aspect of this disclosure, a kit is provided. The kit may contain any of the following components: nucleic acid encoding one or more modified BSH enzymes of this disclosure, and optionally for use with cells transformed or transfected with the nucleic acid, modified BSH enzymes, bacterial cells capable of expressing one or more modified BSH enzymes, pharmaceutical compositions containing one or more BSH enzymes and / or expressing one or more modified BSH enzymes, medical foods containing one or more BSH enzymes and / or expressing one or more modified BSH enzymes, etc., and may optionally contain instructions for performing any of the disclosed methods.

[0133] Pharmaceutical Composition

[0134] In one aspect of this disclosure, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical composition comprises engineered bacterial cells of this disclosure capable of expressing one or more modified BSH enzymes. In some embodiments, the pharmaceutical composition comprises one or more modified BSH enzymes of this disclosure.

[0135] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier or excipient, and its formulation is conventional and readily understood by those skilled in the art.

[0136] method

[0137] In one aspect of this disclosure, a method is provided. In some embodiments, the method includes administering a pharmaceutical composition of this disclosure to a subject in need, the pharmaceutical composition comprising one or more modified BSH enzymes described herein and / or one or more cells expressing modified BSH enzymes.

[0138] As used herein, “subject in need” or “subject” can refer to vertebrates such as livestock (e.g., cows, chickens, goats, sheep, llamas, alpacas, camels, horses, donkeys, fish, crustaceans, mollusks, etc.), humans, companion animals (e.g., dogs, cats, hamsters, rats, chinchillas, ferrets, etc.), and laboratory animals (e.g., mice, rats, rabbits, monkeys, apes, etc.). In some implementations, the subject is a human. In some implementations, the subject is a cat. A subject in need can also refer to a person suffering from NASH, NAFLD, Alzheimer's disease, Crohn's disease, cholestatic disease, or a disease or condition associated with metabolic syndrome.

[0139] The relative abundance of *Christensenella minuta* bacteria in the human gut is positively correlated with a lean host phenotype associated with low BMI. See, for example, Ang, W. et al. A Keystone Gut Bacterium Christensenella minuta—APotential Biotherapeutic Agent for Obesity and Associated Metabolic Diseases. Foods. 2023 Jul; 12(13): 2485; and Mazier, W. et al. A New Strain of Christensenella minuta as a Potential Biotherapy for Obesity and Associated Metabolic Diseases, Cells. 2021 Apr; 10(4): 823; which are incorporated herein by reference in their entirety. In addition, modified *Christensenella minuta* bacteria (NCT04663139) have been studied as a probiotic.

[0140] Fatty liver disease, also known as hepatic steatosis, typically occurs in cats due to prolonged anorexia or starvation. When cats stop eating for extended periods, their bodies mobilize fat reserves to meet energy needs, leading to excessive fat accumulation in the liver. This buildup interferes with liver function, impairing its ability to perform essential tasks such as detoxification and metabolism. Without timely intervention, fatty liver disease can progress rapidly, leading to severe liver dysfunction, jaundice, and potential liver failure. Treatment usually involves aggressive nutritional support, including force-feeding or tube feeding to ensure adequate calorie intake. Veterinary care may also include supportive measures such as fluid therapy, vitamin supplementation, and pharmacological treatment to support liver function. Despite treatment efforts, the prognosis for cats with hepatic steatosis can vary depending on the severity of the disease and the presence of underlying conditions. While many cats recover with timely intervention, a significant proportion may die from complications associated with advanced liver disease. Prevention, early identification, and intervention are crucial for improving outcomes and minimizing the risk of death in cats with fatty liver disease. It is believed that the disclosed modified enzymes, as pharmaceutical compositions in food or probiotics, can treat the signs or symptoms of feline hepatic steatosis.

[0141] In some embodiments, the method is a method of treating non-alcoholic fatty liver disease (NAFLD) in a subject in need, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of this disclosure to the subject to treat NAFLD.

[0142] As used herein, "therapeutic effective amount" or "effective amount" means the amount or dose of a pharmaceutical composition that provides the desired effect in a subject under diagnosis or treatment after a single or multiple administrations of the same dose. For example, a therapeutically effective amount of a pharmaceutical composition of this disclosure may comprise an amount that effectively improves one or more signs or symptoms associated with: for example, NAFLD, non-alcoholic steatohepatitis (NASH), liver cancer such as hepatocellular carcinoma (HCC), Alzheimer's disease, Crohn's disease, hyperlipidemia, dyslipidemia, hypercholesterolemia, obesity, cardiovascular diseases such as coronary artery disease, peripheral artery disease, carotid artery disease, heart failure, and stroke.

[0143] Christensenellaminuta has been reported to be absent from the microbiome of patients with Crohn's disease (CD) and has been documented to induce anti-inflammatory effects in human epithelial cells, supporting its potential as a negative regulator or treatment of CD. See, for example, Relizani, K. et al. Selection of a novel strain of Christensenellaminuta as a future biotherapy for Crohn's disease. Sci Rep. 2022; 12: 6017, which is incorporated herein by reference.

[0144] The disclosed pharmaceutical composition can be used to treat cholestatic diseases such as primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), Alagille syndrome, progressive familial intrahepatic cholestasis (PFIC), or biliary atresia.

[0145] Cholestatic diseases are a group of conditions characterized by impaired bile flow in the liver, leading to the accumulation of bile acids and other substances in the liver and bloodstream. Bile acids are essential for the digestion and absorption of fats and fat-soluble vitamins in the intestines. In cholestatic diseases, bile acid dysfunction occurs due to the disruption of normal bile flow. This dysfunction can lead to a variety of symptoms, including jaundice, itching, and fatigue, and in severe cases, liver damage.

[0146] Bile salt hydrolase (BSH) is an enzyme that plays a crucial role in bile acid metabolism. It catalyzes the breakdown of bile salts into bile acids and amino acids. In cholestatic diseases where bile acids accumulate, BSH can serve as a therapeutic target. By enhancing BSH activity, excess bile salts can be broken down into bile acids, thereby reducing their toxicity to hepatocytes and improving bile flow. This mechanism helps alleviate symptoms associated with cholestatic diseases and may even prevent further liver damage. Therefore, using novel BSH with higher activity as a therapeutic intervention holds promise for treating cholestatic diseases by restoring bile acid homeostasis and promoting liver health. See, for example, Li, T. and Apte, U. Bile acidmetabolism and signaling in cholestasis, inflammation and cancer AdvPharmacol. 2015; 74: 263–302 and Zheng, J. et al. Bile acid-mediated signaling in cholestatic liver diseases Cell & Bioscience volume 13, Article number: 77(2023), each incorporated herein by reference in its entirety.

[0147] Examples of cholestatic diseases can be treated using the disclosed pharmaceutical compositions, probiotics, prebiotics, and methods:

[0148] 1. Primary biliary cholangitis (PBC): This is an autoimmune disease that primarily affects adults, especially middle-aged women. It causes inflammation and destruction of the small bile ducts within the liver.

[0149] 2. Primary sclerosing cholangitis (PSC): Another autoimmune disease, PSC causes inflammation and scarring (fibrosis) of the bile ducts, leading to obstruction and impaired bile flow. PSC can occur in both adults and children.

[0150] 3. Biliary atresia: This is a rare disease affecting newborns and infants. It involves complete or partial obstruction of the intrahepatic and extrahepatic bile ducts, leading to bile accumulation, liver damage, and eventually cirrhosis if left untreated.

[0151] 4. Alagille syndrome: This genetic disorder affects multiple organs, including the liver. Alagille syndrome is characterized by bile duct abnormalities, leading to cholestasis and liver damage. It can occur in infancy or childhood.

[0152] 5. Progressive Familial Intrahepatic Cholestasis (PFIC): PFIC comprises a group of rare, inherited disorders that cause impaired bile formation and cholestasis. It typically manifests in infancy or early childhood and can lead to progressive liver damage.

[0153] 6. Benign recurrent intrahepatic cholestasis (BRIC): BRIC is another rare genetic disorder characterized by intermittent episodes of cholestasis and jaundice. It usually occurs in late childhood or adulthood and is generally less severe than PFIC.

[0154] As used herein, “treatment” and its grammatical variations refer to reducing or preventing at least one sign or symptom of a disease or condition. For example, treating NAFLD may include reducing or preventing weakness, loss of appetite, nausea, jaundice, itching, swelling and edema of the legs and abdomen, confusion, or gastrointestinal (GI) bleeding. In the case of HCC, treatment may refer to reducing abdominal discomfort or bloating, jaundice, gastrointestinal bleeding, nausea or vomiting, persistent itching, or fever. In the case of obesity, treatment may refer to reducing body fat percentage / composition. In the case of hyperlipidemia and hypercholesterolemia, treatment may refer to normalizing the blood lipid or cholesterol profile, respectively. In the case of Alzheimer's disease (AD), treatment may refer to reducing confusion, aphasia, or other AD-related symptoms. In the case of Crohn's disease, treatment may refer to reducing bowel movement frequency, reducing bowel movement-related pain, and gaining weight. In the context of cardiovascular disease, treatment may refer to reducing numbness (especially on one side of the body) in the face, arms or legs, confusion, difficulty speaking or understanding speech; difficulty with vision in one or both eyes, difficulty walking, dizziness and / or loss of balance or coordination.

[0155] In some embodiments, the method is a method of treating non-alcoholic steatohepatitis (NASH) in a subject in need, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of this disclosure to the subject to treat NASH.

[0156] In some embodiments, the method is a method of treating liver cancer in a subject in need, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of this disclosure to the subject to treat liver cancer.

[0157] In some embodiments, the method is a method of treating Alzheimer's disease in a subject in need, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of this disclosure to the subject to treat Alzheimer's disease.

[0158] liver cancer

[0159] The disclosed engineered BSH enzyme has the potential to be utilized as a therapeutic target for liver cancer due to its effects on bile acid metabolism and the gut-hepatic axis. It can be manipulated to disrupt pro-cancer processes and promote anti-cancer mechanisms in the liver. The balance between bound and unbound bile acids is crucial in liver cancer; excessive bound bile acids can promote inflammation and carcinogenesis, while high levels of bound bile acids can lead to hepatocellular damage and promote carcinogenesis. The disclosed enzyme promotes an increase in unbound bile acids, which is a therapeutic benefit for both treating and preventing liver cancer.

[0160] Alzheimer's disease

[0161] The role of lipid metabolism in Alzheimer's disease is an active area of ​​research, and there is evidence that abnormal regulation of lipid metabolism can play a significant role in the development and progression of the disease. Alzheimer's disease is a complex neurodegenerative disease characterized by the accumulation of amyloid plaques and tau tangles in the brain, as well as neuronal cell death and cognitive decline. Cholesterol is a lipid essential for brain function because it is a major component of cell membranes and myelin sheaths (the protective sheaths surrounding nerve fibers). Abnormalities in cholesterol metabolism, such as high levels of LDL (“bad”) cholesterol or low levels of HDL (“good”) cholesterol, have been associated with an increased risk of Alzheimer's disease. It is believed that disruption of cholesterol homeostasis can lead to the accumulation of amyloid-β plaques, a hallmark of Alzheimer's disease.

[0162] Lipid peroxidation (oxidative degradation of lipids) can lead to the production of reactive oxygen species and inflammation. Oxidative stress and inflammation are thought to contribute to the pathogenesis of Alzheimer's disease. Lipid peroxidation can damage cell membranes and impair neuronal function. Lipid rafts are specialized regions of the cell membrane rich in cholesterol and certain types of lipids. They play a role in the processing of amyloid precursor protein (APP), which is cleaved to form amyloid-β, a peptide that accumulates in Alzheimer's disease. Alterations in lipid rafts can affect APP cleavage, potentially leading to increased amyloid-β production.

[0163] APOE is a protein involved in lipid transport in the brain. Different subtypes of APOE exist, and the APOE ε4 allele is a major genetic risk factor for late-onset Alzheimer's disease. This allele has been associated with altered lipid metabolism in the brain and can affect the clearance of amyloid-β from the brain. The composition of lipids in the brain can affect membrane integrity and fluidity, which in turn affects neuronal function. Changes in the levels of specific lipids (such as sphingolipids and phospholipids) have been observed in the brains of individuals with Alzheimer's disease.

[0164] In some embodiments, the method is a method of treating a disease or condition associated with metabolic syndrome in a subject in need, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of this disclosure to the subject to treat the disease or condition associated with metabolic syndrome. In some embodiments, the disease or condition associated with metabolic syndrome is selected from the group consisting of: hyperlipidemia, hypercholesterolemia, obesity, and cardiovascular disease. In some embodiments, the cardiovascular disease is selected from the group consisting of: coronary artery disease, peripheral artery disease, carotid artery disease, heart failure, and stroke.

[0165] As used in this article, “disease or condition associated with metabolic syndrome” means any disease, condition, or finding associated with metabolic syndrome. “Metabolic syndrome” refers to a group of conditions that increase the risk of heart disease, stroke, and diabetes. Metabolic syndrome includes high blood pressure, high blood sugar, excess body fat around the waist, and abnormal cholesterol levels.

[0166] The disclosed compositions and methods can be used to treat signs or symptoms of diabetes, such as type 2 diabetes, including, for example, reducing fasting blood glucose.

[0167] Bioremediation

[0168] BSH enzymes can be used in bioremediation processes to help break down and remove contaminants from water and soil. Some contaminants, such as certain hydrophobic organic compounds, may be difficult to degrade naturally. The modified BSH enzymes disclosed herein can help enhance the degradation of these compounds by altering their interactions with bile salts and microbial communities.

[0169] food

[0170] In one aspect of this disclosure, a food product is provided. In some embodiments, the food product comprises lipids isolated from an organism by the methods of this disclosure, for example, by contacting the organism with a modified enzyme of this disclosure, and further includes isolating, purifying, or extracting the lipids from the organism.

[0171] Food can take any form, such as meat substitutes, food additives, etc.

[0172] Medical food

[0173] In one aspect of this disclosure, a medical food is provided. In some embodiments, the medical food comprises a modified BSH enzyme of this disclosure, a prebiotic, bacterial cells, or probiotics.

[0174] As used herein, “medicinal food” refers to food that is consumed or administered orally under the supervision of a physician and is intended for the specific dietary management of a disease or condition whose specific nutritional requirements are determined based on recognized scientific principles and through medical evaluation.

[0175] Food for medical use may be administered in any form, including but not limited to powders, pills, yogurt, capsules, tablets, gels, etc.

[0176] definition

[0177] The disclosed subject matter may be further described using the following definitions and terms. The definitions and terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0178] As used in this specification and claims, the singular forms “a,” “an,” and “the” include the plural forms unless the context clearly specifies otherwise. For example, unless the context clearly specifies otherwise, the term “substitute” should be interpreted as “one or more substitutions.”

[0179] As used herein, “about,” “approximately,” “substantially,” and “significantly” will be understood by one of ordinary skill in the art and will vary to some extent depending on the specific context in which they are used. If the use of a term is unclear to one of ordinary skill in the art in the context of its use, “about” and “approximately” will mean at most plus or minus 10% of the particular term, and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.

[0180] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising”. The terms “comprise” and “comprising” should be interpreted as “open-ended” transitional terms that allow for the further inclusion of additional components into those components described in the claims. The terms “consist” and “consisting of” should be interpreted as “closed-ended” transitional terms that do not allow for the inclusion of additional components other than those described in the claims. The term “substantially constitutes” should be interpreted as partially closed and only allows for the inclusion of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0181] The phrase “such as” should be interpreted as “for example, including”. Furthermore, the use of any and all exemplary language, including but not limited to “such as”, is intended only to better illustrate the invention and does not constitute a limitation on the scope of the invention, unless otherwise stated.

[0182] Furthermore, in cases where conventions such as "at least one of A, B, and C" are used, this structure is generally intended to make the meaning of the convention understandable to those skilled in the art (e.g., "a system having at least one of A, B, and C" will include, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together). Those skilled in the art should also understand that virtually any transition words and / or phrases representing two or more alternative terms, whether in the specification or drawings, should be understood to include the possibility of including one of the terms, any one of the two terms, or both terms. For example, the phrase "A or B" will be understood to include the possibility of including "A" or "B" or "A and B".

[0183] All words such as “at most,” “at least,” “greater than,” and “less than” include the listed numbers and refer to a range that can be further subdivided into ranges and subranges. A range includes each individual member. Thus, for example, a group with 1-3 members means a group with 1, 2, or 3 members. Similarly, a group with 6 members means a group with 1, 2, 3, 4, or 6 members, and so on.

[0184] The modal verb "may" refers to the preferred use or selection of one or more options or choices among several described implementation schemes or features included therein. Where no options or choices regarding a particular implementation scheme or feature included therein are disclosed, the modal verb "may" refers to an affirmative action regarding how to make or use an aspect of the described implementation scheme or feature included therein, or a clear decision regarding the use of a specific skill regarding the described implementation scheme or feature included therein. In the latter case, the modal verb "may" has the same meaning and connotation as the auxiliary verb "can".

[0185] Explanatory Implementation Plan

[0186] 1. A recombinant bacterial cell, wherein the recombinant bacterial cell carries a heterologous BSH gene from the genome of *Christsenium spp.*

[0187] 2. A recombinant bacterial cell from embodiment 1, wherein the expression of the BSH gene is controlled by a heterologous promoter.

[0188] 3. Engineered bacteria from embodiments 1 and 2, wherein the promoter is an inducible promoter.

[0189] 4. Engineered bacteria from embodiments 1 and 2, wherein the promoter can be induced by environmental conditions, including those specific to the mammalian gut.

[0190] 5. Engineered bacteria from embodiments 1-4, wherein the promoter responds to hypoxic or anaerobic conditions.

[0191] 6. Engineered bacteria from embodiments 1-2, wherein the promoter is a constitutively active promoter.

[0192] 7. Engineered bacteria from embodiments 1-6, further comprising a heterologous gene sequence encoding one or more BSH genes, including DSM33407.

[0193] 8. Engineered bacterial cells from embodiments 1-7, wherein the BSH gene is directly or indirectly linked to an inducible promoter.

[0194] 9. Engineered bacteria from embodiments 1-7, wherein the promoters are induced by environmental factors, including those present in the gut of mammals.

[0195] 10. Engineered bacteria from embodiments 1-7, wherein the promoter can be induced by hypoxia or anaerobic conditions.

[0196] 11. Engineered bacteria from embodiments 1-7, wherein the promoter is a constitutive promoter.

[0197] 12. Engineered bacteria from embodiments 1-7, containing variants of the said gene, which have been further engineered to obtain increased enzyme function, protein stability, broader substrate specificity, or any combination of these characteristics.

[0198] 13. Engineered bacteria from embodiments 1-7, wherein the bacteria have been engineered to contain a combination of at least one BSH gene originally derived from *Christsenia minora* and a second BSH allele from another bacterium commonly found in the gut microbiota of mammals. These microbial communities are associated with bacterial strains including, but not limited to, certain species of *Aminococcus*, certain species of *Actinomyces*, *Akkermansia spp.*, *Akkermansia myxophila*, certain species of *Isobacterium*, certain species of *Anaerobic Cocci*, certain species of *Anaerobic Bacilli*, certain species of *Bacteroides*, other species of *Bacteroides*, *Bacteroides oxytocinoides*, *Bacteroides foetida*, *Bacteroides fragilis*, *Bacteroides ovatus*, *Bacteroides monomorpha*, certain species of *Pasteurella*, *Bifidobacterium adolescentis*, other species of *Bifidobacterium*, certain species of *Bifidobacterium*, certain species of *Biliobacterium*, *Brutella ovatus*, *Brutella brevicula*, other species of *Brutella*, certain species of *Brutella*, certain species of *Brutella*, certain species of *Streptococcus*, certain species of *Christensenella*, and certain species of *Christensenella minuta*.spp), certain species of Citrobacter, certain species of Clostridium, other species of Clostridium, certain species of Clostridium, Clostridium perfringens, certain species of Clostridium, other species of Clostridium, Collins aerogenes, certain species of Collins, Collins faecium, Cladosporium davidii, certain species of Coccidia, certain species of Coccidia, certain species of Coccidia, certain species of Rhodotorulaceae, certain species of Desulfovibrioceae, certain species of Alisteria dilatata, Dorperella formateriate, certain species of Dorperella, other species of Dorperella, Egretella tarda, other species of Enterobacteriaceae, certain species of Enterobacteriaceae, certain species of Enterococcus, certain species of Erysipelothrix, Amyloliquefaciens, Amyloliquefaciens, Amyloliquefaciens, E. longum Certain species of *Eubacterium*, *Clostridium*, *Fusobacterium*, *Gymnococci*, *Haemophilus parainfluenzae*, other species of *Haemophilus*, certain species of *Helicobacter*, other species of *Helicobacter* from the Trichophytonceae family, certain species of Trichophytonceae, *Lactobacillus reuteri*, *Lactobacillus mucosa*, *Lactobacillus zeatus*, certain species of *Lactobacillus*, certain species of *Lactobacillus* from the Lactobacillusceae family, certain species of *Lactococcus*, certain species of *Leuconostoc* family, certain species of *Methanobacterium*, certain species of *Macrococcus*, certain species of *Bacillus methanogenus*, *Gymnococcus*, certain species of *Gymnococcus*, *Syntrophus serratus*, certain species of *Aureobacterium*, certain species of *Vibrio*, *Pseudomonas digiri*, *Pseudomonas*. Certain species, certain species of *Peptococcus*, certain species of the *Peptococcus* family, certain species of *Micromonas*, certain species of *Pediococcus*, other species of *Pediococcus*, certain species of *Peptococcus*, certain species of *Peptococcus*, anaerobic *Streptococcus*, other species of *Streptococcus*, certain species of *Koala*, *Prevotella*, certain species of *Prevotella*, *Stichoprevotella*, *Prevotella* family, certain species of *Proteus*, certain species of *Rikenaceae*, *Roseobacter faecium*, certain species of *Roseobacter*, other species of *Ruminococcus* family, *Ruminococcus brucelli*, active *Ruminococcus*, certain species of *Ruminococcus*, other species of *Ruminococcus*. Species, *Streptococcus truncatula*, certain species of *Slackella*, certain species of S24-7, certain species of SMB53, *Streptococcus pharyngis*, *Streptococcus luteus*, certain species of *Streptococcus*, other species of *Streptococcus*, certain species of *Sartreus*, certain species of *Bacillus zurichae*, *Bleedingella* (UC), *Enterobacteraceae*, *Femobacterium*, *Parabacterium*, *Pediococcus*, certain species of *Curvobacterium*, certain species of *Veillonella*, *Sartreus*, *Bacillus zurichae*, *Clostridium*, *Erysipelothrix*, *Ruminococci*, *Veillonella spp.*, *Veillonella spp.*, and *Weissella*.

[0199] 14. Engineered bacterial cells from embodiments 1-13, wherein the BSH gene is derived from the Kristensenella spp. gene and has been further engineered using synthetic biology tools to improve the protein’s half-life, enzyme activity per unit of protein, bile salt substrate specificity, or any combination of the three characteristics listed herein.

[0200] 15. Engineered bacterial cells from embodiments 1-14, wherein the BSH gene is directly or indirectly linked to an inducible promoter.

[0201] 16. Engineered bacteria from embodiments 1-14, wherein the promoters are induced by environmental factors, including those present in the gut of mammals (humans and / or non-humans).

[0202] 17. Engineered bacteria from embodiments 1-14, wherein the promoter can be induced by hypoxia or anaerobic conditions.

[0203] 18. Engineered bacteria from embodiments 1-14, wherein the promoter is a constitutive promoter.

[0204] 19. Engineered bacteria from embodiments 1-14, containing variants of the said gene, which have been further engineered to obtain increased enzyme function, protein stability, broader substrate specificity, or any combination of these characteristics.

[0205] 20. Engineered bacteria from embodiments 1-19, wherein the heterologous BSH contains genes originally derived from *Christsenium spp.*

[0206] 21. Engineered bacteria from embodiments 1-20, wherein the BSH gene is located in a plasmid within the bacterial cell.

[0207] 22. Engineered bacteria from embodiments 1-20, wherein the BSH gene is integrated into the chromosome of the bacterial cell.

[0208] 23. Engineered bacterial cells from embodiments 1-22, wherein the engineered bacterial cells are members of the genera Bacteroides, Bifidobacterium, Clostridium, Escherichia coli, Lactobacillus, Lactococcus, or Zurich bacillus.

[0209] 24. A pharmaceutical composition comprising engineered bacterial cells of any one of embodiments 1-23 and a pharmaceutically acceptable carrier.

[0210] 25. A method for treating a disease associated with bile salt regulation disorder in a subject in need, the method comprising administering a pharmaceutical composition of embodiment 24 to the subject.

[0211] 26. A method for reducing bile salt levels in the intestine of a subject, the method comprising administering a pharmaceutical composition of embodiment 24 to the subject, thereby reducing bile salt levels in the subject's intestine.

[0212] 27. A method for reducing the amount of conjugated bile salts, unconjugated bile salts, or both conjugated and unconjugated bile salts in a subject, the method comprising administering a pharmaceutical composition of embodiment 24 to the subject, thereby reducing the amount of conjugated bile salts, unconjugated bile salts, or both conjugated and unconjugated bile salts in the intestine of the subject.

[0213] 28. The method of implementation scheme 25, wherein the condition associated with bile salt regulation disorder is a metabolic disease or a liver fat storage disease.

[0214] 29. The method of implementation scheme 25, wherein the condition associated with bile salt regulation disorder is cardiovascular disease.

[0215] 30. A method for improving clinical signs or symptoms in mammals exhibiting NAFLD, the method comprising administering bacterial cells containing a BSH gene derived from Kristensenia minora strain DSM33407, or a functional equivalent of the gene having at least 90% identity with the BSH gene derived from Kristensenia minora strain DSM33407.

[0216] 31. The method of embodiment 30, wherein the bacterial cells express the BSH gene after being introduced into the mammalian intestine.

[0217] 32. The method of any one of embodiments 25-31, wherein the bacterial cells are administered as part of a formulation suitable for oral delivery.

[0218] 33. The method of any one of embodiments 1-32, wherein the subject is a mammal.

[0219] 34. The method of implementation scheme 33, wherein the subject is a human being.

[0220] 35. The method of implementation scheme 33, wherein the subject is a dog, cat, horse, ferret, guinea pig, or hamster.

[0221] 36. The method of implementation scheme 33, wherein the subject is a cow, goat, sheep, deer, yak, pig, donkey, reindeer, llama, alpaca, camel, or rabbit.

[0222] 37. The method of embodiment 30, wherein the bacterial cell is engineered Christensenella pyrenoidosa.

[0223] 38. A method for producing improved animal products, the method comprising administering a pharmaceutical composition of embodiment 24 to livestock animals; harvesting said livestock animals or products derived from said livestock animals to produce improved animal products.

[0224] 39. The method of any one of Implementation Schemes 25-38, wherein the administration includes intravenous, intramuscular, subcutaneous, rectal, vaginal or oral administration.

[0225] 40. The method of implementation scheme 39, wherein administration includes oral or rectal administration.

[0226] 41. The method of implementation scheme 40, wherein administration includes oral administration.

[0227] Example

[0228] The following examples are illustrative and should not be construed as limiting the scope of the claimed subject matter.

[0229] Example 1 - BSH Expression

[0230] introduction

[0231] Purpose

[0232] The purpose of this protocol is to describe a complete experimental setup from E. coli culture to the detection of products following the enzymatic reaction of cell lysates with substrates.

[0233] Background Information

[0234] Previous experiments confirmed the effectiveness of using 100 µM IPTG in OD. 600 Induction at ~1.0 and subsequent expression at 37°C for 20 hours resulted in BSH expression.

[0235] Material

[0236] chemicals

[0237] Isopropyl β-d-1-thiogalactoside (CAS: 367-93-1)

[0238] Buffer / culture medium

[0239]

[0240] Consumables: Microtiter plates, 2.2mL square V-bottom deep-well plates

[0241] Laboratory equipment: flatbed shaker, centrifuge, spectrometer

[0242] method

[0243] Day 1 (Pre-culture)

[0244] Prepare a microtiter plate containing 145 µL of LB medium with 50 µg / mL kanamycin. Thaw the cryogenic culture of the variant (BSH variant of Escherichia coli C41 pLys). After resuspending, inoculate 5 μL of the variant into each well. Seal the plate with 150 μL of MTP. Incubate the MTP at 37 °C with shaking at 900 rpm for 16 hours.

[0245] Day 2 (Main Training)

[0246] To assess cell growth, 5 μL of the preculture was diluted 1:40 in 195 μL of LB medium. Absorption was measured at 600 nm. The preculture should be diluted in LB to OD0.05. 600 ~0.05 was used for the master culture. 495 μL of LB containing 50 μg / mL kanamycin was added to each well of the deep-well plate. 5 μL of pre-culture was added to the culture medium. The inoculated medium was grown at 37°C, 900 rpm, and 80% humidity with shaking until the bacterial culture reached OD500. 600 ~1.0, therefore OD is measured during the culture process. 600 Once OD is reached 600 ~1.0, cells were induced with 5 μL of 10 mM IPTG to a final concentration of 100 μM IPTG. After induction, DWPs were sealed with breathable foil. Expression was performed for 20 hours at 37°C, 900 rpm, and 80% humidity with shaking.

[0247] Harvest

[0248] After 20 hours of expression, the plates were centrifuged at 4,000g for 10 minutes at 4°C to harvest the cells. The supernatant was then discarded. The cell pellet was frozen at -20°C for at least 1 hour.

[0249] Example 2 - Taurine Determination

[0250] introduction

[0251] Purpose

[0252] The aim of this predictive experiment is to determine the taurine concentration following a BSH assay of TCA. Taurine concentration indicates BSH activity. See, for example, Figure 2 The following protocols can be used to detect the activity of the disclosed BSH enzyme in the hydrolysis of, for example, taurocholic acid (TCA) to produce taurine and cholic acid.

[0253] Background Information

[0254] The sample from Example 1 was used.

[0255] Material

[0256] chemicals

[0257] Taurine Assay Kit (MET-5071, Cell Biolabs)

[0258] Buffer / culture medium

[0259]

[0260] Consumables: Transparent MTP F-base.

[0261] Laboratory equipment: ELISA reader, plate shaker

[0262] method

[0263] Detailed Plan

[0264] Preparation for Taurine Determination

[0265] Thaw the sample from the BSH assay. Thaw the taurine standard and taurine assay buffer from the taurine assay kit at room temperature. Thaw the remaining components on ice. Prepare standards in duplicate at 0 μM, 15.6 μM, 31.3 μM, 62.5 μM, 125 μM, 250 μM, 500 μM, and 1000 μM. Prepare the reaction mixture according to the handbook, see Table 3.

[0266] Table 3: Reaction mixture of the taurine assay kit.

[0267]

[0268] Sample preparation

[0269] On one hand, 50 μL of deproteinized BSH sample (pH 7) was transferred to MTP. On the other hand, 33 μL of deproteinized BSH sample (pH 5) was transferred to MTP, and 17 μL of 200 mM TRIS / HCl buffer (pH 9) was added to these samples. Adding pH 9 buffer to the pH 5 sample is necessary to achieve a pH of ~7. All samples were measured repeatedly. One replicate was measured directly using the taurine kit, while the other replicate underwent quenching to determine sample background (see next section – Taurine Assay).

[0270] Taurine determination

[0271] Transfer 50 μL of the standard to MTP. Initiate the reaction with 50 μL of the reaction mixture. Incubate at 25°C with shaking for 30 minutes. Terminate the reaction with 50 μL of stop solution and 50 μL of developer. To detect sample background, quench the reaction with 50 μL of quenching solution and 50 μL of developer, and incubate at 25°C with shaking for 3 minutes. Absorbance (415 nm) is measured using a microplate reader. Subtract the absorbance value of the sample background (i.e., quenching) from the absorbance value of the sample terminated with the stop solution.

[0272] Example 3 - Glycine Determination

[0273] introduction

[0274] Background Information

[0275] Thaw the sample from the BSH assay (Example 1). Thaw the glycine standard and glycine assay buffer from the glycine assay kit at room temperature. Thaw the remaining components on ice. Prepare standards in duplicate at 0 μM, 1.56 μM, 3.13 μM, 6.25 μM, 12.5 μM, 25.0 μM, 50.0 μM, and 100.0 μM. Prepare the reaction mixture according to the manual.

[0276] Material

[0277] chemicals

[0278] Glycine Assay Kit (ab211100, abcam)

[0279]

[0280] Consumables: Black MTP; Laboratory equipment: Microplate reader, plate shaker.

[0281] method

[0282] Thaw the samples. Thaw the components of the glycine assay kit at room temperature with GLY assay buffer, and thaw the remaining components of the kit on ice. Prepare a 1 mM standard by diluting 5 μL of 100 mM GLY standard with 495 μL ddH2O. Prepare a 50 μM standard by diluting 50 μL of 1 mM GLY standard with 950 μL ddH2O. Use 0 μM, 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM glycine as standards. Prepare the reaction mixture according to the handbook for one reaction, see Table 5.

[0283] Table 5: Reaction mixture of the glycine assay kit.

[0284]

[0285] Sample preparation

[0286] Dilute the pH 7 sample 1:3 in the assay buffer. Dilute the pH 5 sample 2:3 in the pH 9 buffer, and then 1:2 in the assay buffer.

[0287] Glycine assay

[0288] Transfer 50 μL of the standard to black MTP. Transfer 50 μL of the 1:3 diluted sample to black MTP. Initiate the reaction with 50 μL of the reaction mixture. Incubate in the dark at 37°C and 900 rpm with shaking for 30–60 minutes. Measure fluorescence at Ex / Em 560 / 587 after 60 minutes.

[0289] Example 4 - Treatment of non-alcoholic fatty liver disease

[0290] In one embodiment, a subject with nonalcoholic fatty liver disease (NAFLD) is administered a therapeutically effective amount of the disclosed composition, such as a pharmaceutical composition, prebiotic, or probiotic. The composition may be administered via any route indicated by the subject's specific treatment needs, such as oral or rectal administration. Administration of the composition may reduce the signs and symptoms of NAFLD. Treatment may be administered daily, every other day, every three days, or according to a schedule determined by the patient's progress, as determined by the physician. Compared to untreated subjects, subjects are expected to experience an increase in weight, appetite, or relief of nausea, vomiting, abdominal pain, itching, or other indicators associated with the reduction of signs or symptoms of NAFLD. Methods for measuring the reduction of signs and symptoms of NAFLD are known in the art.

[0291] Example 5 - Treatment of non-alcoholic steatohepatitis

[0292] In one embodiment, a subject with nonalcoholic steatohepatitis (NASH) is administered a therapeutically effective amount of the disclosed composition, such as a pharmaceutical composition, prebiotic, or probiotic. The composition may be administered via any route indicated by the subject's specific treatment needs, such as oral or rectal administration. Administration of the composition may reduce the signs and symptoms of NASH. Treatment may be administered daily, every other day, every three days, or according to a schedule determined by the patient's progress, as determined by the physician. Compared to untreated subjects, subjects are expected to experience an increase in weight, appetite, or relief of nausea, vomiting, abdominal pain, itching, or other indicators associated with the reduction of signs or symptoms of NASH. Methods for measuring the reduction of signs and symptoms of NASH are known in the art.

[0293] Table 6. Polynucleotide sequences encoding the disclosed modified enzymes.

[0294]

[0295] In the foregoing description, it will be apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention described illustratively herein can be suitably practiced without any one or more elements or limitations not specifically disclosed herein. Terms and expressions used herein are descriptive rather than limiting, and their use is not intended to exclude any equivalents of the shown and described features or portions thereof, but rather to recognize that various modifications are possible within the scope of the invention. Therefore, it should be understood that although the invention has been shown by way of specific embodiments and optional features, modifications and / or variations of the concepts disclosed herein can be adopted by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention.

[0296] This document may cite multiple patent and non-patent references. All cited references are incorporated herein by reference in their entirety. In the event of any discrepancy between the definition of a term in this specification and its definition in the cited references, the definition in this specification shall prevail.

Claims

1. A modified Christensenella minuta (C. minuta) bile salt hydrolase (BSH) enzyme, which is at least 90% identical to SEQ ID NO: 1, and contains one or more amino acid substitutions at positions C2, Y8, Y34, N46, H47, Y56, I57, Y64, P81, K90, F99, L127, S136, Y176, K189, S206, R207, A221, N292 or R323 relative to SEQ ID NO:

1.

2. The modified enzyme according to claim 1, wherein the one or more substitutions include C2S, Y8K, Y8T, Y8Q, Y8V, Y8T, Y34N, Y34S, N46K, H47S, H47N, Y56A, Y56M, Y56T, Y56L, Y56V, I57V, Y64S, Y64F, P81A, K90E, F99Y, L127I, L127V, S136A, S136T, Y176K, K189P, K189N, S206G, R207Q, A221P, A221L, N292S, N292R, N292H, R323P, R323T, R323E, R323L, or R323Q.

3. The modified enzyme according to claim 1, wherein one or more substitutions include Y8V, Y34K, I57V, Y64S, P81A, K90E, L127I, S136A, K189P, K189N, S206G, R207Q, N292S, R323L or R323E.

4. The modified enzyme according to claim 1, wherein the one or more substitutions include I57V.

5. The modified enzyme according to claim 1, wherein one or more substitutions include Y64S.

6. The modified enzyme according to claim 1, wherein one or more substitutions include S136A.

7. The modified enzyme according to claim 1, wherein the one or more substitutions include K189P.

8. The modified enzyme according to claim 1, wherein the one or more substitutions include Y8V, P81A, S206G and R207Q.

9. The modified enzyme according to claim 1, wherein the one or more substitutions include Y34K, I57V, L127I and N292S.

10. The modified enzyme according to claim 1, wherein the one or more substitutions include Y56T, I57V, L127I and N292S.

11. The modified enzyme according to claim 1, wherein one or more substitutions include K90E, L127I, N292S and R323E.

12. The modified enzyme according to claim 1, wherein the one or more substitutions include Y34N, N46K, H47N, Y64F, Y176K and A221L.

13. The modified enzyme according to claim 1, wherein one or more substitutions include Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S and R323L.

14. The modified enzyme according to claim 1, wherein the modified enzyme comprises one of SEQ ID NO: 2-47.

15. The modified enzyme of claim 1, wherein the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 5, and wherein the modified enzyme has a substituted I57V relative to SEQ ID NO: 1, optionally, wherein the modified enzyme comprises or is composed of SEQ ID NO:

5.

16. The modified enzyme of claim 1, wherein the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 6, and wherein the modified enzyme has a substituted Y64S relative to SEQ ID NO: 1, optionally, wherein the modified enzyme comprises or is composed of SEQ ID NO:

6.

17. The modified enzyme of claim 1, wherein the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 8, and wherein the modified enzyme has a substituted S136A relative to SEQ ID NO: 1, optionally, wherein the modified enzyme comprises or is composed of SEQ ID NO:

8.

18. The modified enzyme of claim 1, wherein the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 9, and wherein the modified enzyme has a substituted K189P relative to SEQ ID NO: 1, optionally, wherein the modified enzyme comprises or is composed of SEQ ID NO:

9.

19. The modified enzyme of claim 1, wherein the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 36, and wherein the modified enzyme has substitutions of Y8V, P81A, S206G and R207Q relative to SEQ ID NO: 1, optionally, wherein the modified enzyme comprises or is composed of SEQ ID NO:

36.

20. The modified enzyme of claim 1, wherein the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 37, and wherein the modified enzyme has substitutions of Y34K, I57V, L127I and N292S relative to SEQ ID NO: 1, optionally, wherein the modified enzyme comprises or is composed of SEQ ID NO:

37.

21. The modified enzyme of claim 1, wherein the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 41, and wherein the modified enzyme has substitutions of Y56T, I57V, L127I and N292S relative to SEQ ID NO: 1, optionally, wherein the modified enzyme comprises or is composed of SEQ ID NO:

41.

22. The modified enzyme of claim 1, wherein the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 42, and wherein the modified enzyme has substitutions of K90E, L127I, N292S and R323E relative to SEQ ID NO: 1, optionally, wherein the modified enzyme comprises or is composed of SEQ ID NO:

42.

23. The modified enzyme of claim 1, wherein the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 43, and wherein the modified enzyme has substitutions of Y34N, H47N, Y64F, Y176K, A221P relative to SEQ ID NO: 1; optionally, wherein the modified enzyme comprises or is composed of SEQ ID NO:

43.

24. The modified enzyme of claim 1, wherein the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 44, and wherein the modified enzyme has substitutions of Y34N, N46K, H47N, Y64F, Y176K and A221L relative to SEQ ID NO: 1, optionally, wherein the modified enzyme comprises or is composed of SEQ ID NO:

44.

25. The modified enzyme of claim 1, wherein the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 45, and wherein the modified enzyme has substitutions of Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S and R323L relative to SEQ ID NO: 1, optionally, wherein the modified enzyme comprises or is composed of SEQ ID NO:

45.

26. A modified bile salt hydrolase (BSH) enzyme comprising amino acid substitutions at the following positions relative to SEQ ID NO: 1: I5V, Y8R, T9S, D11H, H12T, Y13F, R16M, L18Y, L20A, E21S, F22S, Y24E, N25G, T27K, V28I, T29V, V30I, T31V, K33R, Y34K, F37L, H38K, F39L, R40S, K43E, L45I, N46S, H47N, M54I, Y56K, I57V, V58I, D60N, F61Y, Y64F, Y65K, D66M, T68C, S74A. G77A, L78I, N79S, D82G, N83I, D85S, K87G, V89P, K90T, E91P, Y93K, D94E, I96V, F99Y, F101L, W104Y, Q108R, A110G, S113D, R11 6L, I117E, E120K, Q121T, I122L, L124I, L127V, N128D, E131A, L133H, S136K, L138V, H139K, W140Y, Q145K, R146E, D147K, V151L , S153Q, F161Y, V165T, N170G, F174Y, M178L, K189N, E190K, T195K, A197S, A198P, E199S, L200I, E201K, Q204R, Y205I, R207S, A211N, L218Y, S220D, A221L, R223K, V225I, K226R, A228S, T230V, K231R, M232L, S234A, E240Y, S241D, S243Q, I244L, S245M, G2 52D, E255R, Q256N, Q257V, R258P, C260T, E265G, E269L, I270R, I272L, S274Q, S275T, C277I, N278D, K281R, Y284L, T287R, E290N, N292T, Y300N, E302C, N303D, D305N, N307D, T308K, S311E, Y312F, M315V, K316T, Q318P, N321Y, Y322E, R323L, N324K, and Y325K.

27. A modified bile salt hydrolase (BSH) enzyme comprising amino acid substitutions at the following positions relative to SEQ ID NO: 1: I5V, Y8K, T9S, D11S, H12S, Y13W, R16M, L18Y, L20A, E21S, F22S, Y24E, T27E, V28I, T29V, V30I, T31V, K33R, Y34K, F37L, H38K, F39L, R40S, K43T, L45I, N46D, H47E, Y49N, M54I, Y56E, V58R, D60N, L 63I, Y64F, Y65K, D66M, G77A, L78V, N79S, F80L, D82G, N83I, D85S, K87G, V89K, K90T, Y93K, D94E, F99Y, F10 1L, W104Y, Q108R, C109A, A110S, S113E, R116L, I117K, Q121N, I122L, L124I, L127V, N128D, L133R, S136K, L138V, H139R, W140F, S143A, Q145K, R146D, D147K, S148A, S153Q, F161Y, V165T, N170G, T173L, F174Y, M178L, K189N, E190A, T195K, A198P, E199N, L 200I, E201K, Q204R, Y205I, R207A, M209D, A211N, L218K, S220D, A221L, R2 23K, V225I, K226R, A228T, T230V, K231R, M232L, S234A, S239T, S241D, S24 3Q, I244L, S245M, G252D, E255R, Q256D, Q257V, R258P, C260T, H262W, E265G, E269L, I270R, I272L, S274Q, S275V, C276A, C277I, K281T, I283K, Y284L, T287R, E290N, N292S, T295N, A296M, Y300N, E302C, N303D, D305N, G306S, N307D, T308K, Y312F, M315Q, Q318L, Q319D, N321K, Y322E, R323L, and Y325K.

28. A modified bile salt hydrolase (BSH) enzyme comprising or consisting of one of SEQ ID NO: 46 or 47.

29. The modified enzyme according to claim 1, wherein the modified enzyme has increased activity compared to wild-type Christensenella pyrenoidosa BSH at about pH 5.

30. The modified enzyme according to claim 1, wherein the modified enzyme has increased activity compared to wild-type Christensenella pyrenoidosa BSH at approximately pH 7.

31. A polynucleotide comprising a sequence encoding a modified Christensenella pyrenoidosa bile salt hydrolase (BSH) enzyme as described in claim 1.

32. The polynucleotide of claim 31, wherein the polynucleotide comprises one of SEQ ID NO: 48-79.

33. The polynucleotide of claim 31, further comprising one or more regulatory elements, wherein the one or more regulatory elements are operatively linked to a sequence encoding the modified enzyme.

34. The polynucleotide of claim 31, wherein the one or more regulatory elements comprise a promoter and / or an enhancer.

35. The polynucleotide of claim 31, further comprising a selection marker.

36. The polynucleotide of claim 33, wherein the one or more regulatory elements are promoters, and the promoters are constitutive promoters.

37. The polynucleotide of claim 33, wherein the one or more regulatory elements are promoters, and the promoter is an inductive promoter.

38. The polynucleotide of claim 37, wherein the promoter is induced by environmental conditions or in response to hypoxia or anaerobic conditions.

39. The polynucleotide of claim 37, wherein the polynucleotide comprises one of SEQ ID NO: 104-109.

40. A prebiotic comprising the modified Christensenella pyrenoidosa bile salt hydrolase (BSH) enzyme as described in claim 1.

41. A pharmaceutical composition comprising the modified Christensenella pyrenoidosa bile salt hydrolase (BSH) enzyme according to claim 1.

42. An engineered bacterial cell comprising the polynucleotide according to claim 31.

43. The engineered bacterial cell of claim 42, wherein the expression of said polynucleotide is operatively linked to an exogenous promoter not found in the natural Christensenella pyrenoidosa genome.

44. The engineered bacterial cell according to claim 42, wherein the engineered bacterial cell is selected from the group consisting of: certain species of *Aminococcus*, certain species of *Actinomyces*, *Ackermania*, certain species of *Isobacterium*, certain species of *Anaerobic Cocci*, certain species of *Anaerobic Bacilli*, certain species of *Bacteroides*, certain species of *Bacteroides*, other species of *Bacteroides*, *Bacteroides acidogenicus*, *Bacteroides foetida*, *Bacteroides fragilis*, *Bacteroides ovatus*, *Bacteroides monomorpha*, certain species of *Pasteurella*, *Bifidobacterium adolescentis*, other species of *Bifidobacterium*, certain species of *Bifidobacterium*, certain species of *Biliobacterium*, *Brutella ovatus*, *Brutella brevicula*, other species of *Brutella*, certain species of *Brutella*, certain species of *Brutella*, certain species of *Streptococcus*, and certain species of *Christsenia*. Certain species of *Citrobacter*, certain species of *Closporia*, other species of *Closporales*, certain species of *Closporales*, *Clostridium perfringens*, certain species of *Closporium*, other species of *Closporium*, *Colinsella vesiculosus*, certain species of *Colinsella faecium*, *Clostridium davidii*, certain species of *C. davidii*, certain species of *C. davidii*, certain species of *Rhodotorula*, certain species of *Desulfovibrioceae*, certain species of *D. dialliste*, *D. formateriel*, certain species of *D. davidii*, other species of *D. davidii*, *Eragrostis stenosis*, other species of *Enterobacteria*, certain species of *Enterobacteria*, certain species of *Enterococcus*, certain species of *Erysipelothrix*, *Amycosis rubra*, *Amycosis stenosis*, *Amycosis rubra*, *Amycosis stenosis*, certain species of *E. davidii*, *Amycosis stenosis*, certain species of *E. davidii*, *C. davidii*, certain species of *C. davidii*, certain species of *C. davidii*, certain species of *C. davidii*, *Amycosis stenosis*, *Amycosis stenosis*, *Haemophilus parainfluenzae*, other species of *Haemophilus*, snails Certain species of *Bacillus*, other species of *Helicobacter* (family Trichophyceae), certain species of *Trichophyceae*, *Lactobacillus reuteri*, *Lactobacillus mucosa*, *Lactobacillus zeatus*, certain species of *Lactobacillus*, certain species of *Lactobacillus* (family Lactobacillus), certain species of *Lactococcus*, certain species of *Leuconostoc* (family Leuconostocaceae), certain species of *Macromonas*, certain species of *Macrococcus*, certain species of *Methanobacterium*, *Gymnospermum*, certain species of *Gymnospermum*, *Syntrophus schrei*, certain species of *Aureobacterium*, certain species of *Vibrio*, *Peptococcus*, certain species of *Peptostreptococcus*, anaerobic *Streptococcus*, other species of *Streptococcus*, certain species of *Koala*. Prevotella, certain species of Prevotella, Prevotella stearacea, Prevotellaceae, Proteus, certain species of Rikenaceae, Rosette, certain species of Rosette, other species of Ruminococci, certain species of Ruminococciceae, Ruminococcus brucelli, active Ruminococcus, certain species of Ruminococcus, other species of Ruminococcus, Ruminococcus tortiformis, certain species of Slackella, certain species of S24-7, certain species of SMB53, Streptococcus pharyngitis, Streptococcus luteus, certain species of Streptococcus, other species of Streptococcus, certain species of Sartella, certain species of Bacillus zurichense, UC Breederella, UC Enterobacteriaceae, UC Faecalibacterium, UC Parabacterium, UC Pediococcus, certain species of Curvularia, certain species of Veillonella.*Sartreus* spp., *Zurichobacterium* spp., *UC* Clostridium orders, *UC* Erysipelothrix family, *UC* Ruminococci family, *Veillonella* spp., certain species of *Veillonella* spp., *Veillonella* spp., and *Weissella* spp.

45. The bacterial cell of claim 42, wherein the polynucleotide is integrated into the genome of the bacterial cell.

46. ​​A probiotic composition comprising engineered bacterial cells according to claim 42.

47. A pharmaceutical composition comprising the engineered bacterial cells according to claim 42 or the modified BSH enzyme according to claim 1.

48. A method comprising administering the pharmaceutical composition of claim 47 to a subject in need.

49. The method of claim 48, wherein the subject in need suffers from one or more of the following: non-alcoholic fatty liver disease (NAFLD), a disease or condition associated with metabolic syndrome, or cardiovascular disease.

50. A method of treating non-alcoholic fatty liver disease (NAFLD) in a subject in need, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 47 to the subject to treat NAFLD.

51. A method of treating non-alcoholic steatohepatitis (NASH) in a subject in need, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 47 to the subject to treat NASH.

52. A method of treating liver cancer in a subject in need, the method comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 47 to the subject to treat liver cancer.

53. A method of treating cholestatic disease in a subject in need, the method comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 47 to the subject to treat the cholestatic disease in the subject.

54. A method of treating Alzheimer's disease in a subject in need, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 47 to the subject to treat Alzheimer's disease.

55. A method of treating Crohn's disease in a subject in need, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 47 to the subject to treat Crohn's disease in the subject.

56. A method of treating a disease or condition associated with metabolic syndrome in a subject in need, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 47 to the subject to treat the disease or condition associated with metabolic syndrome.

57. The method of claim 47, wherein the metabolic syndrome-related disease or condition is selected from the group consisting of: hyperlipidemia, hypercholesterolemia, obesity, and cardiovascular disease.

58. The method of claim 57, wherein the cardiovascular disease is selected from the group consisting of: coronary artery disease, peripheral artery disease, carotid artery disease, heart failure, and stroke.

59. The method according to any one of claims 48-58, wherein the desired subject is an animal.

60. The method of claim 59, wherein the desired subject is a mammal.

61. The method of claim 59, wherein the desired subject is a dog, cat, cow, sheep, goat, chicken, turkey, pig, fish, crustacean, or mollusc.

62. The method of claim 59, wherein the subject in need is a human subject.

63. The method of claim 59, wherein the desired subject is a cat, and wherein the cat suffers from feline hepatic steatosis.

64. A method comprising delivering a probiotic according to claim 46 or a prebiotic according to claim 40 to a subject.

65. A method for improving livestock productivity, the method comprising providing an effective amount of the probiotic according to claim 46 to improve livestock productivity.

66. The method of claim 65, wherein the livestock yield is improved compared to members of the same species of livestock that are not provided with the probiotic.

67. The method of claim 66, wherein the livestock is a ruminant, pig, bird, fish, crustacean, or mollusc.

68. The method of claim 67, wherein the ruminant is selected from dairy cows, sheep, goats, deer, buffalo, or camels.

69. The food product according to claim 68, wherein the lipid is a fatty acid.

70. A medical food comprising the modified BSH enzyme according to claim 1, the prebiotic according to claim 40, or the bacterial cells according to claim 42.

71. A method for preparing a modified bile salt hydrolase (BSH) enzyme, the method comprising introducing the polynucleotide according to claim 31 into a cell, thereby causing the cell to express the modified BSH enzyme.

72. The method of claim 71, further comprising enriching, purifying, or separating the modified BSH enzyme.

73. A modified BSH enzyme of *Scleroderma pulvinatum*, having at least 90% identity with SEQ ID NO: 1, wherein the modified BSH enzyme has at least one substitution modification relative to SEQ ID NO: 1.

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