Modified bile salt hydrolase enzyme and method of use thereof

Abstract

Modified Christensenella minuta bile salt hydrolase (BSH) enzyme, prebiotics containing the same, engineered bacterial cells containing the modified BSH enzyme, probiotics containing engineered bacterial cells, methods of using compositions containing the modified enzyme for treating diseases and disorders, and foods are disclosed.

Description

Technical Field

[0001] (Cross - reference to Related Applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 493,219, filed on March 30, 2023, the entire content of which is incorporated herein by reference.

[0002] Sequence Listing The sequence listing is submitted as an xml file named "179136_00009.xml", created on March 29, 2024, attached to this application and having a size of 116,308 bytes. The sequence listing is submitted electronically via Patent Center, and the entire content of which is incorporated herein by reference.

[0003] The field of the present disclosure relates to a modified bile salt hydrolase enzyme, a composition comprising the modified enzyme, and methods of using the enzyme.

Background Art

[0004] Bile salt hydrolase (BSH) enzymes that catalyze the deconjugation of bile acids play an important role, inter alia, in the absorption of dietary fat and the regulation of the microbiome. Furthermore, the functions of BSH enzymes are associated with human and animal diseases. Therefore, there is a need for modified BSH enzymes in the art.

Summary of the Invention

[0005] Modified Christensenella minuta bile salt hydrolase enzyme One aspect of the present disclosure provides a modified Christensenella minuta bile salt hydrolase (BSH) enzyme. In some embodiments, the modified enzyme is at least 90% identical to SEQ ID NO: 1 and includes 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, one or more substitutions 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 includes or consists of one of sequence numbers 2 to 47. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with sequence number 5, the modified enzyme has the substitution I57V for sequence number 1, and optionally, the modified enzyme includes or consists of sequence number 5. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with sequence number 6, the modified enzyme has the substitution Y64S for sequence number 1, and optionally, the modified enzyme includes or consists of sequence number 6. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 8, and the modified enzyme has the substitution S136A for SEQ ID NO: 1, and optionally the modified enzyme includes or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 9, and the modified enzyme has the substitution K189P for SEQ ID NO: 1, and optionally the modified enzyme includes or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 36, and the modified enzyme has the substitutions Y8V, P81A, S206G, and R207Q for SEQ ID NO: 1, and optionally the modified enzyme includes 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 the modified enzyme has substitutions Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1, and 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 the modified enzyme has substitutions Y56T, I57V, L127I, and N292S relative to SEQ ID NO: 1, and optionally, the modified enzyme comprises or consists of SEQ ID NO: 41.In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 42, and the modified enzyme has substitutions K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 42. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 43, and the modified enzyme has substitutions Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 43. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 44, and the modified enzyme has substitutions Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 44. In some embodiments, the modified enzyme contains a sequence having at least 90% identity with SEQ ID NO: 45, and the modified enzyme has substitutions Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1, and optionally, the modified enzyme contains or consists of SEQ ID NO: 45. In some embodiments, the modified enzyme has increased activity compared to wild-type C. minuta BSH at approximately pH 5. In some embodiments, the modified enzyme has increased activity compared to wild-type C. minuta BSH at approximately pH 7. In some embodiments, the modified Christensenella minuta bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, and the modified BSH enzyme has at least one substitution modification relative to SEQ ID NO: 1.

[0006] In one aspect of this disclosure, a modified BSH enzyme is provided. In some embodiments, the modified bile salt hydrolase (BSH) enzyme is located at position 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 relative to SEQ ID NO: 1 , Y65K, D66M, T68C, S74A, G77A, L78I, N79S, D82G, N83I, D85S, K87G, V89P, K90T, E91P, Y93K, D94E, I96V, F99Y, F101L, W104Y, Q108R, A110G, S113D, R116L, I117E, E120K, Q121T, I122L, L124I, L127V, N128D, E131A, L133H, S136K, L138V, H139K, W140Y, Q145K, R146E, D 147K, V151L, S153Q, F161Y, V165T, N170G, F174Y, M178L, K189N, E190K, T195K, A197S, A198P, E199S, L200I, E201K, Q204R, Y205I, R2 07S, A211N, L218Y, S220D, A221L, R223K, V225I, K226R, A228S, T230V, K231R, M232L, S234A, E240Y, S241D, S243Q, I244L, S245M, G25 The amino acid substitutions include 2D, 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) enzymes are located at positions 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, L63I, Y64F, Y65K, relative to SEQ ID NO: 1 D66M, G77A, L78V, N79S, F80L, D82G, N83I, D85S, K87G, V89K, K90T, Y93K, D94E, F99Y, F101L, W104Y, Q108R, C109A, A110S, S113E, R11 6L, 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, L200I, E201K, Q204R, Y205I, R207A, M209D, A211N, L218K, S220D, A221L, R223K, V225I, K226R, A228T, T230V, K231R, M232L, S234A, S239T, S241D, S243Q, I244L, S245M, G252D, E255R, Q256D, Q2 The amino acid substitutions include 57V, 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 NOs: 46 or 47.

[0007] Polynucleotides In one aspect of the present disclosure, a polynucleotide is provided. In some embodiments, the polynucleotide comprises a sequence encoding a modified Christensenella minuta bile salt hydrolase (BSH) enzyme, which is at least 90% identical to SEQ ID NO: 1 and includes 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 includes 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, one or more substitutions 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 includes or consists of one of sequence numbers 2 to 47. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with sequence number 5, the modified enzyme has substitution I57V for sequence number 1, and optionally, the modified enzyme includes or consists of sequence number 5. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 6, and the modified enzyme has the substitution Y64S for SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 6. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 8, and the modified enzyme has the substitution S136A for SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 9, and the modified enzyme has the substitution K189P for SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 36, and the modified enzyme has the substitutions Y8V, P81A, S206G, and R207Q for SEQ ID NO: 1, and optionally, the modified enzyme includes 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 the modified enzyme has substitutions Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1, and optionally, the modified enzyme comprises or consists of SEQ ID NO: 37.In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 41, and the modified enzyme has substitutions Y56T, I57V, L127I, and N292S relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 41. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 42, and the modified enzyme has substitutions K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 42. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 43, and the modified enzyme has substitutions Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 43. In some embodiments, the modified enzyme contains a sequence having at least 90% identity with SEQ ID NO: 44, and the modified enzyme has substitutions Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1, and optionally, the modified enzyme contains or consists of SEQ ID NO: 44. In some embodiments, the modified enzyme contains a sequence having at least 90% identity with SEQ ID NO: 45, and the modified enzyme has substitutions Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1, and optionally, the modified enzyme contains or consists of SEQ ID NO: 45. In some embodiments, the modified enzyme has increased activity at approximately pH 5 compared to wild-type C. minuta BSH. In some embodiments, the modified enzyme has increased activity at approximately pH 7 compared to wild-type C. minuta BSH. In some embodiments, the modified Christensenella minuta bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, and 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, which are operably linked to a sequence encoding the modified enzyme.In some embodiments, one or more regulatory elements include a promoter and / or enhancer. In some embodiments, the polynucleotide further includes 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 inductive promoters. In some embodiments, the promoters are inductive to environmental conditions or responsive to hypoxic or anaerobic conditions. In some embodiments, the polynucleotide includes one of SEQ ID NOs: 48-79.

[0008] Prebiotics In one aspect of the present disclosure, a prebiotic is provided. In some embodiments, the prebiotic comprises a modified Christensenella minuta bile salt hydrolase (BSH) enzyme that is at least 90% identical to SEQ ID NO: 1 and includes 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, one or more substitutions 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 includes or consists of one of sequence numbers 2 to 47. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with sequence number 5, the modified enzyme has the substitution I57V for sequence number 1, and optionally, the modified enzyme includes or consists of sequence number 5. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with sequence number 6, the modified enzyme has the substitution Y64S for sequence number 1, and optionally, the modified enzyme includes or consists of sequence number 6. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 8, and the modified enzyme has the substitution S136A for SEQ ID NO: 1, and optionally the modified enzyme includes or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 9, and the modified enzyme has the substitution K189P for SEQ ID NO: 1, and optionally the modified enzyme includes or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 36, and the modified enzyme has the substitutions Y8V, P81A, S206G, and R207Q for SEQ ID NO: 1, and optionally the modified enzyme includes 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 the modified enzyme has substitutions Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1, and 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 the modified enzyme has substitutions Y56T, I57V, L127I, and N292S relative to SEQ ID NO: 1, and optionally, the modified enzyme comprises or consists of SEQ ID NO: 41.In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 42, and the modified enzyme has substitutions K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 42. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 43, and the modified enzyme has substitutions Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 43. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 44, and the modified enzyme has substitutions Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 44. In some embodiments, the modified enzyme contains a sequence having at least 90% identity with SEQ ID NO: 45, and the modified enzyme has substitutions Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1, and optionally, the modified enzyme contains or consists of SEQ ID NO: 45. In some embodiments, the modified enzyme has increased activity compared to wild-type C. minuta BSH at approximately pH 5. In some embodiments, the modified enzyme has increased activity compared to wild-type C. minuta BSH at approximately pH 7. In some embodiments, the modified Christensenella minuta bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, and the modified BSH enzyme has at least one substitution modification relative to SEQ ID NO: 1.

[0009] Pharmaceutical composition containing modified BSH enzyme In one aspect of the present disclosure, a pharmaceutical composition is provided. In some embodiments, the pharmaceutical composition comprises a modified critensenella minuta bile salt hydrolase (BSH) enzyme that is at least 90% identical to SEQ ID NO: 1 and includes 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, one or more substitutions 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 includes or consists of one of sequence numbers 2 to 47. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with sequence number 5, the modified enzyme has the substitution I57V for sequence number 1, and optionally, the modified enzyme includes or consists of sequence number 5. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with sequence number 6, the modified enzyme has the substitution Y64S for sequence number 1, and optionally, the modified enzyme includes or consists of sequence number 6. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 8, and the modified enzyme has the substitution S136A for SEQ ID NO: 1, and optionally the modified enzyme includes or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 9, and the modified enzyme has the substitution K189P for SEQ ID NO: 1, and optionally the modified enzyme includes or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 36, and the modified enzyme has the substitutions Y8V, P81A, S206G, and R207Q for SEQ ID NO: 1, and optionally the modified enzyme includes 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 the modified enzyme has substitutions Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1, and 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 the modified enzyme has substitutions Y56T, I57V, L127I, and N292S relative to SEQ ID NO: 1, and optionally, the modified enzyme comprises or consists of SEQ ID NO: 41.In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 42, and the modified enzyme has substitutions K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 42. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 43, and the modified enzyme has substitutions Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 43. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 44, and the modified enzyme has substitutions Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 44. In some embodiments, the modified enzyme contains a sequence having at least 90% identity with SEQ ID NO: 45, and the modified enzyme has substitutions Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1, and optionally, the modified enzyme contains or consists of SEQ ID NO: 45. In some embodiments, the modified enzyme has increased activity compared to wild-type C. minuta BSH at approximately pH 5. In some embodiments, the modified enzyme has increased activity compared to wild-type C. minuta BSH at approximately pH 7. In some embodiments, the modified Christensenella minuta bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, and the modified BSH enzyme has at least one substitution modification relative to SEQ ID NO: 1.

[0010] Manipulated bacterial cells In one aspect of the present disclosure, an engineered bacterial cell is provided. In some embodiments, the engineered bacterial cell comprises a polynucleotide comprising a sequence encoding a modified Christensenella minuta bile salt hydrolase (BSH) enzyme, which is at least 90% identical to SEQ ID NO: 1 and includes 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 includes 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, one or more substitutions 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 are included. 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 includes or consists of one of sequence numbers 2 to 47. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with sequence number 5, the modified enzyme has substitution I57V for sequence number 1, and optionally, the modified enzyme includes or consists of sequence number 5. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 6, and the modified enzyme has the substitution Y64S for SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 6. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 8, and the modified enzyme has the substitution S136A for SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 9, and the modified enzyme has the substitution K189P for SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 36, and the modified enzyme has the substitutions Y8V, P81A, S206G, and R207Q for SEQ ID NO: 1, and optionally, the modified enzyme includes 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 the modified enzyme has substitutions Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1, and optionally, the modified enzyme comprises or consists of SEQ ID NO: 37. In some embodiments,The modified enzyme contains a sequence having at least 90% identity with SEQ ID NO: 41, and the modified enzyme has substitutions Y56T, I57V, L127I, and N292S relative to SEQ ID NO: 1, and optionally the modified enzyme contains or consists of SEQ ID NO: 41. In some embodiments, the modified enzyme contains a sequence having at least 90% identity with SEQ ID NO: 42, and the modified enzyme has substitutions K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1, and optionally the modified enzyme contains or consists of SEQ ID NO: 42. In some embodiments, the modified enzyme contains a sequence having at least 90% identity with SEQ ID NO: 43, and the modified enzyme has substitutions Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1, and optionally the modified enzyme contains or consists of SEQ ID NO: 43. In some embodiments, the modified enzyme contains a sequence having at least 90% identity with SEQ ID NO: 44, and the modified enzyme has substitutions Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1, and optionally, the modified enzyme contains or consists of SEQ ID NO: 44. In some embodiments, the modified enzyme contains a sequence having at least 90% identity with SEQ ID NO: 45, and the modified enzyme has substitutions Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1, and optionally, the modified enzyme contains or consists of SEQ ID NO: 45. In some embodiments, the modified enzyme has increased activity at approximately pH 5 compared to wild-type C. minuta BSH. In some embodiments, the modified enzyme has increased activity at approximately pH 7 compared to wild-type C. minuta BSH. In some embodiments, the modified Christensenella minuta bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, and 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, the regulatory elements being operably linked to the sequence encoding the modified enzyme. In some embodiments,One or more regulatory elements include a promoter and / or an enhancer. In some embodiments, the polynucleotide further includes 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 inductive promoters. In some embodiments, the promoters are inductive to environmental conditions or responsive to hypoxic or anaerobic conditions. In some embodiments, the polynucleotide includes one of SEQ ID NOs: 48-79. In some embodiments, the expression of the polynucleotide is functionally related to an exogenous promoter not found in the natural Christensenella minuta genome. In some embodiments, the manipulated bacterial cells include those of the genera Acidaminococcus, Actinomyces, Ackermansia muciniphylla, Alobaculum, Anaerococcus, Anaerostipes, Bacteroides, Bacteroides others, Bacteroides acidifaciens, Bacteroides coprofilus, Bacteroides fragilis, Bacteroides ovatus, Bacteroides uniformis, Valnesiella, Bifidobacterium adolescentis, Bifidobacterium others, Bifidobacterium, Bilophila, Brautia obeum, Brautia producta, Brautia others, Brautia, Burraydia, Catenibacterium, and Chrysenella. , Citrobacter genus, Clostridaceae genus, Clostridiales other, Clostridiales genus, Clostridium perfringens, Clostridium genus, Clostridium other, Chorinthera aerofasciens, Chorinthera genus, Chorinthera stercolis, Coprococcus catus, Coprococcus genus, Coriobacterium seae genus, Desulfovibryon genus, Dialist genus, Dorea formisigenerans, Dorea genus, Dorea other, Aegatacera lenta, Enterobacteria seae other, Enterobacteria seae genus, Enterococcus genus, Erysipelotrichaceae genus, Eubacterium biforme, Eubacterium biforme, Eubacterium doricam, Eubacterium genus,Faecalibacterium prausnitzii, Fusobacterium genus, Gemeraceae genus, Haemophilus parainfluenzae, Haemophilus and others, Helicobacter genus, Helicobacter lachnospiraceae and others, Lachnospiraceae genus, Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus zeae, Lactobacillus genus, Lactobacillus lachnospiraceae genus, Lactococcus genus, Leuconostocaceae genus, Megamonas genus, Megasphere genus, Metanobrevibacter genus, Mitsuokera maltafida, Mitsuokera genus, Musispirium shedrelii, Odolibacter genus, Osirospira genus, Parabacteroides distasonis, Parabacter Genus Roydes, Paraprevotella, Paraprevotellaceae, Parvimonas, Pediococcus, Pediococcus and others, Peptococcus, Peptoniphyllus, Peptostreptococcus anaerobius, Peptostreptococcus and others, Phascolarctobacterium, Prevotella copri, Prevotella, Prevotella stercorea, Prevotellaceae, Proteus, Lyceneraceae, Rosebria fesis, Rosebria, Ruminococcusceae and others, Ruminococcus, Ruminococcus bromii, Ruminococcus gunavas, Ruminococcus, Ruminococcus and others, Ruminococcus turkes, Slacchia, S24-7 The group is selected from spp., SMB53 spp., Streptococcus anginosus, Streptococcus lutesiae, Streptococcus genus, other Streptococcus species, Stellera genus, Tulicibacter genus, UC Braidia, UC Enterobacteria seae, UC Faecalibacterium, UC Parabacteroides, UC Pediococcus, Baribaculum genus, Veillonella genus, Sattala, Tulicibacter, UC Clostridial, UC Erysipelotrichacea, UC Luminococcea, Veillonella parbra, Veillonella genus, Veillonella dispa, and Weissella genus. In some embodiments, polynucleotides are incorporated into the genome of the bacterial cell.

[0011] Probiotics In one aspect of the present disclosure, a probiotic is provided. In some embodiments, the probiotic comprises a polynucleotide-based engineered bacterial cell comprising a sequence encoding a modified Christensenella minuta bile salt hydrolase (BSH) enzyme that is at least 90% identical to SEQ ID NO: 1 and includes 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 includes 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, one or more substitutions 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 includeThis includes 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 includes or consists of one of sequence numbers 2 to 47. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 5, and the modified enzyme has the substitution I57V for SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 5. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 6, and the modified enzyme has the substitution Y64S for SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 6. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 8, and the modified enzyme has the substitution S136A for SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 9, and the modified enzyme has the substitution K189P for SEQ ID NO: 1, and optionally, the modified enzyme includes 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 the modified enzyme has substitutions Y8V, P81A, S206G, and R207Q relative to SEQ ID NO: 1, and 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 the modified enzyme has substitutions Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1, and optionally, the modified enzyme comprises or consists of SEQ ID NO: 37.or consisting thereof. In some embodiments, the modified enzyme comprises a sequence having at least 90% identity with SEQ ID NO: 41, and the modified enzyme has substitutions Y56T, I57V, L127I, and N292S relative to SEQ ID NO: 1, and optionally, the modified enzyme comprises or consists 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 the modified enzyme has substitutions K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1, and optionally, the modified enzyme comprises or consists 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 the modified enzyme has substitutions Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1, and optionally, the modified enzyme comprises or consists of SEQ ID NO: 43. In some embodiments, the modified enzyme contains a sequence having at least 90% identity with SEQ ID NO: 44, and the modified enzyme has substitutions Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1, and optionally, the modified enzyme contains or consists of SEQ ID NO: 44. In some embodiments, the modified enzyme contains a sequence having at least 90% identity with SEQ ID NO: 45, and the modified enzyme has substitutions Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1, and optionally, the modified enzyme contains or consists of SEQ ID NO: 45. In some embodiments, the modified enzyme has increased activity at approximately pH 5 compared to wild-type C. minuta BSH. In some embodiments, the modified enzyme has increased activity at approximately pH 7 compared to wild-type C. minuta BSH. In some embodiments, the modified Christensenella minuta bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, and 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,The modified enzyme is operably ligated to the sequence encoding the modified enzyme. In some embodiments, one or more regulatory elements include a promoter and / or enhancer. In some embodiments, the polynucleotide further includes 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 inductive promoters. In some embodiments, the promoters are inductive to environmental conditions or responsive to hypoxic or anaerobic conditions. In some embodiments, the polynucleotide includes one of sequence numbers 48-79. In some embodiments, the expression of the polynucleotide is functionally related to an exogenous promoter not found in the natural Christensenella minuta genome. In some embodiments, the manipulated bacterial cells include those of the genera Acidaminococcus, Actinomyces, Ackermansia muciniphylla, Alobaculum, Anaerococcus, Anaerostipes, Bacteroides, Bacteroides others, Bacteroides acidifaciens, Bacteroides coprofilus, Bacteroides fragilis, Bacteroides ovatus, Bacteroides uniformis, Valnesiellaceae, Bifidobacterium adolescentis, Bifidobacterium others, Bifidobacterium, Bilophila, Brautia obeum, Brautia producta, Brautia others, Brautia, and Burraydia. Genus, Catenibacterium, Chrysenella, Citrobacter, Clostridiaceae, Clostridiales and others, Clostridiales and others, Clostridiales and others, Clostridium perfringens, Clostridium, Clostridium and others, Chorinthera aerofasciens, Chorinthera, Chorinthera stercolis, Coprococcus catus, Coprococcus, Coriobacterium seae, Desulfovibrion, Dialist, Dorea formisigenerans, Dorea, Dorea and others, Aegatella lenta, Enterobacterium seae and others, Enterobacterium seae, Enterococcus, Erysipelotrichaseae, Eubacterium biforme,Eubacterium biforme, Eubacterium doricum, Eubacterium genus, Faecalibacterium prausnitzii, Fusobacterium genus, Gemeraceae genus, Haemophilus parainfluenzae, Haemophilus and others, Helicobacter genus, Helicobacter lachnospiraceae and others, Lachnospiraceae genus, Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus zeae, Lactobacillus genus, Lactobacillus genus, Lactococcus genus, Leuconostocaceae genus, Megamonas genus, Megasphere genus, Metanobrevibacter genus, Atractylodes japonica, Atractylodes genus, Musispirium sheldrellii, Odolibacter genus, Osirospira genus, Paraba Cteroides distasonis, Parabacteroides genus, Paraprevotella genus, Paraprevotella family genus, Parvimonas genus, Pediococcus genus, Pediococcus and others, Peptococcus genus, Peptoniphyllus genus, Peptostreptococcus anaerobius, Peptostreptococcus and others, Phascolarctobacterium genus, Prevotella copri, Prevotella genus, Prevotella stercorea, Prevotella family, Proteus genus, Lycenera family, Rosebria fesis, Rosebria genus, Ruminococcus family and others, Ruminococcus genus, Ruminococcus bromii, Ruminococcus gunavas, Ruminococcus genus, Ruminococcus and others, Ruminococcus turkes, Slacchia genus, S24-7 The group is selected from spp., SMB53 spp., Streptococcus anginosus, Streptococcus lutesiae, Streptococcus genus, other Streptococcus species, Stellera genus, Tulicibacter genus, UC Braidia, UC Enterobacteria seae, UC Faecalibacterium, UC Parabacteroides, UC Pediococcus, Baribaculum genus, Veillonella genus, Sattala, Tulicibacter, UC Clostridial, UC Erysipelotrichacea, UC Luminococcea, Veillonella parbra, Veillonella genus, Veillonella dispa, and Weissella genus. In some embodiments, polynucleotides are incorporated into the genome of the bacterial cell.

[0012] Pharmaceutical composition containing manipulated bacterial cells In one aspect of the present disclosure, a pharmaceutical composition is further provided. In some embodiments, the pharmaceutical composition comprises a polynucleotide-based engineered bacterial cell comprising a sequence encoding a modified Christensenella minuta bile salt hydrolase (BSH) enzyme, which is at least 90% identical to SEQ ID NO: 1 and includes 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 includes 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, one or more substitutions 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 are included. 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 includes or consists of one of sequence numbers 2 to 47. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 5, and the modified enzyme has the substitution I57V for SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 5. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 6, and the modified enzyme has the substitution Y64S for SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 6. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 8, and the modified enzyme has the substitution S136A for SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 9, and the modified enzyme has the substitution K189P for SEQ ID NO: 1, and optionally, the modified enzyme includes 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 the modified enzyme has substitutions Y8V, P81A, S206G, and R207Q relative to SEQ ID NO: 1, and 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 the modified enzyme has substitutions Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1,Optionally, the modified enzyme includes or consists of SEQ ID NO: 37. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 41, and the modified enzyme has substitutions Y56T, I57V, L127I, and N292S relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 41. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 42, and the modified enzyme has substitutions K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists 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 the modified enzyme has substitutions Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1, and optionally, the modified enzyme comprises or consists 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 the modified enzyme has substitutions Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1, and optionally, the modified enzyme comprises or consists 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 the modified enzyme has substitutions Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1, and optionally, the modified enzyme comprises or consists of SEQ ID NO: 45. In some embodiments, the modified enzyme has increased activity compared to wild-type C. minuta BSH at approximately pH 5. In some embodiments, the modified enzyme has increased activity compared to wild-type C. minuta BSH at approximately pH 7. In some embodiments, the modified Christensenella minuta bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, and 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.The regulatory element is operably linked to a sequence encoding a modified enzyme. In some embodiments, one or more regulatory elements include a promoter and / or enhancer. In some embodiments, the polynucleotide further includes 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 inductive promoters. In some embodiments, the promoters are inductive to environmental conditions or responsive to hypoxic or anaerobic conditions. In some embodiments, the polynucleotide includes one of sequence numbers 48-79. In some embodiments, the expression of the polynucleotide is functionally related to an exogenous promoter not found in the natural Christensenella minuta genome. In some embodiments, the manipulated bacterial cells include those of the genera Acidaminococcus, Actinomyces, Ackermansia muciniphylla, Alobaculum, Anaerococcus, Anaerostipes, Bacteroides, Bacteroides other, Bacteroides acidifaciens, Bacteroides coprofilus, Bacteroides fragilis, Bacteroides ovatus, Bacteroides uniformis, Valnesiellaceae, Bifidobacterium adolescentis, Bifidobacterium other, Bifidobacterium, Bilophila, Brautia obeum, Brautia producta, Brautia other, and Brautia. , genus Burraydia, genus Catenibacterium, genus Chrysenella, genus Citrobacter, genus Clostridiaceae, other orders Clostridiales, genus Clostridiales, Clostridium perfringens, genus Clostridium, other Clostridium, Chorinthera aerofasciens, genus Chorinthera, genus Chorinthera stercolis, Coprococcus catus, genus Coprococcus, genus Coriobacterium seae, genus Desulfovibrion, genus Dialist, Dorea formisigenerans, genus Dorea, other Dorea, Aegatella lenta, other Enterobacteria seae, genus Enterobacterium seae, genus Enterococcus, genus Erysipelotrichaceae,Eubacterium biforme, Eubacterium biforme, Eubacterium doricum, Eubacterium genus, Faecalibacterium prausnitzii, Fusobacterium genus, Gemeraceae genus, Haemophilus parainfluenzae, Haemophilus and others, Helicobacter genus, Helicobacter lacnospiraceae and others, Lactobacillus genus, Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus zeae, Lactobacillus genus, Lactobacillus genus, Lactococcus genus, Leuconostocaceae genus, Megamonas genus, Megasphere genus, Metanobrevibacter genus, Atractylodes japonica, Atractylodes genus, Musispirium shelderi, Odolibacter genus, Osiros Pyramid genus, Parabacteroides distasonis, Parabacteroides genus, Paraprevotella genus, Paraprevotella family genus, Parvimonas genus, Pediococcus genus, Pediococcus and others, Peptococcus genus, Peptoniphyllus genus, Peptostreptococcus anaerobius, Peptostreptococcus and others, Phascolarctobacterium genus, Prevotella copri, Prevotella genus, Prevotella stercorea, Prevotella family, Proteus genus, Lycenera family, Rosebria fesis, Rosebria genus, Ruminococcus family and others, Ruminococcus genus, Ruminococcus bromii, Ruminococcus gunavas, Ruminococcus genus, Ruminococcus and others, Ruminococcus turkes, Slacchia genus, S24-7 The group is selected from spp., SMB53 spp., Streptococcus anginosus, Streptococcus lutesiae, Streptococcus genus, other Streptococcus species, Stellera genus, Tulicibacter genus, UC Braidia, UC Enterobacteria seae, UC Faecalibacterium, UC Parabacteroides, UC Pediococcus, Baribaculum genus, Veillonella genus, Sattala, Tulicibacter, UC Clostridial, UC Erysipelotrichacea, UC Luminococcea, Veillonella parbra, Veillonella genus, Veillonella dispa, and Weissella genus. In some embodiments, polynucleotides are incorporated into the genome of the bacterial cell.

[0013] method In one aspect of the present disclosure, a method is provided. In some embodiments, the method involves administering to a subject requiring the use of a pharmaceutical composition comprising a modified BSH enzyme that is at least 90% identical to SEQ ID NO: 1 and includes 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, one or more substitutions 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 includes or consists of one of sequence numbers 2 to 47. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with sequence number 5, the modified enzyme has the substitution I57V for sequence number 1, and optionally, the modified enzyme includes or consists of sequence number 5. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with sequence number 6, the modified enzyme has the substitution Y64S for sequence number 1, and optionally, the modified enzyme includes or consists of sequence number 6. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 8, and the modified enzyme has the substitution S136A for SEQ ID NO: 1, and optionally the modified enzyme includes or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 9, and the modified enzyme has the substitution K189P for SEQ ID NO: 1, and optionally the modified enzyme includes or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 36, and the modified enzyme has the substitutions Y8V, P81A, S206G, and R207Q for SEQ ID NO: 1, and optionally the modified enzyme includes 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 the modified enzyme has substitutions Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1, and 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 the modified enzyme has substitutions Y56T, I57V, L127I, and N292S relative to SEQ ID NO: 1, and optionally, the modified enzyme comprises or consists of SEQ ID NO: 41.In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 42, and the modified enzyme has substitutions K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 42. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 43, and the modified enzyme has substitutions Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 43. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 44, and the modified enzyme has substitutions Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 44. In some embodiments, the modified enzyme contains a sequence having at least 90% identity with SEQ ID NO: 45, and the modified enzyme has substitutions Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1, and optionally, the modified enzyme contains or consists of SEQ ID NO: 45. In some embodiments, the modified enzyme has increased activity compared to wild-type C. minuta BSH at approximately pH 5. In some embodiments, the modified enzyme has increased activity compared to wild-type C. minuta BSH at approximately pH 7. In some embodiments, the modified Christensenella minuta bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, and the modified BSH enzyme has at least one substitution modification relative to SEQ ID NO: 1. In some embodiments, the required subjects are those suffering from one or more of the following: non-alcoholic fatty liver disease (NAFLD), diseases or disorders associated with metabolic syndrome, or cardiovascular disease.

[0014] In some embodiments, a method is provided for treating non-alcoholic fatty liver disease (NAFLD) in a subject requiring treatment for NAFLD, comprising administering a therapeutically effective amount of a pharmaceutical composition to the subject, the 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, to treat the NAFLD. In some embodiments, one or more substitutions 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 includes or consists of one of sequence numbers 2 to 47. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with sequence number 5, the modified enzyme has substitution I57V for sequence number 1, and optionally, the modified enzyme includes or consists of sequence number 5. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 6, and the modified enzyme has the substitution Y64S for SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 6. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 8, and the modified enzyme has the substitution S136A for SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 9, and the modified enzyme has the substitution K189P for SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 36, and the modified enzyme has the substitutions Y8V, P81A, S206G, and R207Q for SEQ ID NO: 1, and optionally, the modified enzyme includes 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 the modified enzyme has substitutions Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1, and optionally, the modified enzyme comprises or consists of SEQ ID NO: 37.In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 41, and the modified enzyme has substitutions Y56T, I57V, L127I, and N292S relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 41. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 42, and the modified enzyme has substitutions K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 42. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 43, and the modified enzyme has substitutions Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 43. In some embodiments, the modified enzyme contains a sequence having at least 90% identity with SEQ ID NO: 44, and the modified enzyme has substitutions Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1, and optionally, the modified enzyme contains or consists of SEQ ID NO: 44. In some embodiments, the modified enzyme contains a sequence having at least 90% identity with SEQ ID NO: 45, and the modified enzyme has substitutions Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1, and optionally, the modified enzyme contains or consists of SEQ ID NO: 45. In some embodiments, the modified enzyme has increased activity at approximately pH 5 compared to wild-type C. minuta BSH. In some embodiments, the modified enzyme has increased activity at approximately pH 7 compared to wild-type C. minuta BSH. In some embodiments, the modified Christensenella minuta bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, and the modified BSH enzyme has at least one substitution modification relative to SEQ ID NO: 1.

[0015] In some embodiments, a method is provided for treating non-alcoholic steatohepatitis (NASH) in a subject requiring treatment for NASH, comprising administering a therapeutically effective amount of a pharmaceutical composition to the subject, the 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, to treat NASH. In some embodiments, one or more substitutions 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 includes or consists of one of sequence numbers 2 to 47. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with sequence number 5, the modified enzyme has the substitution I57V for sequence number 1, and optionally, the modified enzyme includes or consists of sequence number 5. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with sequence number 6, the modified enzyme has the substitution Y64S for sequence number 1, and optionally, the modified enzyme includes or consists of sequence number 6. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 8, and the modified enzyme has the substitution S136A for SEQ ID NO: 1, and optionally the modified enzyme includes or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 9, and the modified enzyme has the substitution K189P for SEQ ID NO: 1, and optionally the modified enzyme includes or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 36, and the modified enzyme has the substitutions Y8V, P81A, S206G, and R207Q for SEQ ID NO: 1, and optionally the modified enzyme includes 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 the modified enzyme has substitutions Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1, and 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 the modified enzyme has substitutions Y56T, I57V, L127I, and N292S relative to SEQ ID NO: 1, and optionally, the modified enzyme comprises or consists of SEQ ID NO: 41.In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 42, and the modified enzyme has substitutions K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 42. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 43, and the modified enzyme has substitutions Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 43. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 44, and the modified enzyme has substitutions Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 44. In some embodiments, the modified enzyme contains a sequence having at least 90% identity with SEQ ID NO: 45, and the modified enzyme has substitutions Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1, and optionally, the modified enzyme contains or consists of SEQ ID NO: 45. In some embodiments, the modified enzyme has increased activity compared to wild-type C. minuta BSH at approximately pH 5. In some embodiments, the modified enzyme has increased activity compared to wild-type C. minuta BSH at approximately pH 7. In some embodiments, the modified Christensenella minuta bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, and the modified BSH enzyme has at least one substitution modification relative to SEQ ID NO: 1.

[0016] In some embodiments, a method is provided for treating liver cancer in a subject requiring treatment for liver cancer, comprising administering a therapeutically effective amount of a pharmaceutical composition to the subject, the 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, to treat the liver cancer. In some embodiments, one or more substitutions 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 includes or consists of one of sequence numbers 2 to 47. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with sequence number 5, the modified enzyme has the substitution I57V for sequence number 1, and optionally, the modified enzyme includes or consists of sequence number 5. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with sequence number 6, the modified enzyme has the substitution Y64S for sequence number 1, and optionally, the modified enzyme includes or consists of sequence number 6. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 8, and the modified enzyme has the substitution S136A for SEQ ID NO: 1, and optionally the modified enzyme includes or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 9, and the modified enzyme has the substitution K189P for SEQ ID NO: 1, and optionally the modified enzyme includes or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 36, and the modified enzyme has the substitutions Y8V, P81A, S206G, and R207Q for SEQ ID NO: 1, and optionally the modified enzyme includes 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 the modified enzyme has substitutions Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1, and 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 the modified enzyme has substitutions Y56T, I57V, L127I, and N292S relative to SEQ ID NO: 1, and optionally, the modified enzyme comprises or consists of SEQ ID NO: 41.In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 42, and the modified enzyme has substitutions K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 42. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 43, and the modified enzyme has substitutions Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 43. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 44, and the modified enzyme has substitutions Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 44. In some embodiments, the modified enzyme contains a sequence having at least 90% identity with SEQ ID NO: 45, and the modified enzyme has substitutions Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1, and optionally, the modified enzyme contains or consists of SEQ ID NO: 45. In some embodiments, the modified enzyme has increased activity compared to wild-type C. minuta BSH at approximately pH 5. In some embodiments, the modified enzyme has increased activity compared to wild-type C. minuta BSH at approximately pH 7. In some embodiments, the modified Christensenella minuta bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, and the modified BSH enzyme has at least one substitution modification relative to SEQ ID NO: 1.

[0017] In some embodiments, a method is provided for treating Alzheimer's disease in a subject requiring treatment for Alzheimer's disease, comprising administering a therapeutically effective amount of a pharmaceutical composition to the subject, the 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, to treat Alzheimer's disease. In some embodiments, one or more substitutions 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 includes or consists of one of sequence numbers 2 to 47. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with sequence number 5, the modified enzyme has the substitution I57V for sequence number 1, and optionally, the modified enzyme includes or consists of sequence number 5. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with sequence number 6, the modified enzyme has the substitution Y64S for sequence number 1, and optionally, the modified enzyme includes or consists of sequence number 6. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 8, and the modified enzyme has the substitution S136A for SEQ ID NO: 1, and optionally the modified enzyme includes or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 9, and the modified enzyme has the substitution K189P for SEQ ID NO: 1, and optionally the modified enzyme includes or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 36, and the modified enzyme has the substitutions Y8V, P81A, S206G, and R207Q for SEQ ID NO: 1, and optionally the modified enzyme includes 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 the modified enzyme has substitutions Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1, and 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 the modified enzyme has substitutions Y56T, I57V, L127I, and N292S relative to SEQ ID NO: 1, and optionally, the modified enzyme comprises or consists of SEQ ID NO: 41.In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 42, and the modified enzyme has substitutions K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 42. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 43, and the modified enzyme has substitutions Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 43. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 44, and the modified enzyme has substitutions Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 44. In some embodiments, the modified enzyme contains a sequence having at least 90% identity with SEQ ID NO: 45, and the modified enzyme has substitutions Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1, and optionally, the modified enzyme contains or consists of SEQ ID NO: 45. In some embodiments, the modified enzyme has increased activity compared to wild-type C. minuta BSH at approximately pH 5. In some embodiments, the modified enzyme has increased activity compared to wild-type C. minuta BSH at approximately pH 7. In some embodiments, the modified Christensenella minuta bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, and the modified BSH enzyme has at least one substitution modification relative to SEQ ID NO: 1.

[0018] In some embodiments, a method is provided for treating a disease or disorder related to metabolic syndrome in a subject requiring treatment for such a disease or disorder, comprising administering a therapeutically effective amount of a pharmaceutical composition to the subject, the 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, thereby treating the disease or disorder related to metabolic syndrome. In some embodiments, one or more substitutions 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 includes or consists of one of sequence numbers 2 to 47. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with sequence number 5, the modified enzyme has substitution I57V for sequence number 1, and optionally, the modified enzyme includes or consists of sequence number 5. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 6, and the modified enzyme has the substitution Y64S for SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 6. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 8, and the modified enzyme has the substitution S136A for SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 8. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 9, and the modified enzyme has the substitution K189P for SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 9. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 36, and the modified enzyme has the substitutions Y8V, P81A, S206G, and R207Q for SEQ ID NO: 1, and optionally, the modified enzyme includes 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 the modified enzyme has substitutions Y34K, I57V, L127I, and N292S relative to SEQ ID NO: 1, and optionally, the modified enzyme comprises or consists of SEQ ID NO: 37.In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 41, and the modified enzyme has substitutions Y56T, I57V, L127I, and N292S relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 41. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 42, and the modified enzyme has substitutions K90E, L127I, N292S, and R323E relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 42. In some embodiments, the modified enzyme includes a sequence having at least 90% identity with SEQ ID NO: 43, and the modified enzyme has substitutions Y34N, H47N, Y64F, Y176K, and A221P relative to SEQ ID NO: 1, and optionally, the modified enzyme includes or consists of SEQ ID NO: 43. In some embodiments, the modified enzyme contains a sequence having at least 90% identity with SEQ ID NO: 44, and the modified enzyme has substitutions Y34N, N46K, H47N, Y64F, Y176K, and A221L relative to SEQ ID NO: 1, and optionally, the modified enzyme contains or consists of SEQ ID NO: 44. In some embodiments, the modified enzyme contains a sequence having at least 90% identity with SEQ ID NO: 45, and the modified enzyme has substitutions Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L relative to SEQ ID NO: 1, and optionally, the modified enzyme contains or consists of SEQ ID NO: 45. In some embodiments, the modified enzyme has increased activity at approximately pH 5 compared to wild-type C. minuta BSH. In some embodiments, the modified enzyme has increased activity at approximately pH 7 compared to wild-type C. minuta BSH. In some embodiments, the modified Christensenella minuta bile salt hydrolase (BSH) enzyme has at least 90% identity with SEQ ID NO: 1, and the modified BSH enzyme has at least one substitution modification relative to SEQ ID NO: 1. In some embodiments, the disease or disorder 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.

[0019] In one aspect of the present disclosure, a medical food is provided. In some embodiments, the medical food comprises a modified BSH enzyme of the present disclosure, a prebiotic of the present disclosure, a probiotic of the present disclosure, or a bacterial cell of the present disclosure.

[0020] In one aspect of the present disclosure, a method for producing a modified bile salt hydrolase (BSH) enzyme is provided, the method comprising introducing a polynucleotide of the present disclosure into a cell, thereby expressing the modified BSH enzyme in the cell. In some embodiments, the method further comprises concentrating, purifying, or isolating the modified BSH enzyme. In some embodiments, the cell is a mammalian cell, an insect cell, a fungal cell, or a bacterial cell.

Brief Description of the Drawings

[0021] [Figure 1] Shows an exemplary enzymatic reaction by the disclosed BSH enzyme.

[0022] [Figure 2] Shows an exemplary enzymatic reaction by the disclosed BSH enzyme.

[0023] [Figure 3A] Shows exemplary data demonstrating GCA hydrolysis and glycine detection by the disclosed enzyme. [Figure 3B] Shows exemplary data demonstrating GCA hydrolysis and glycine detection by the disclosed enzyme.

[0024] [Figure 4A] Shows exemplary data demonstrating GCDCA hydrolysis and glycine detection by the disclosed enzyme. [Figure 4B] Shows exemplary data demonstrating GCDCA hydrolysis and glycine detection by the disclosed enzyme.

[0025] [Figure 5A] An exemplary polyacrylamide gel demonstrating the successful expression of the modified BSH enzyme is shown. Staining: Fixed in staining solution (25% (v / v) isopropanol, 10% (v / v) acetic acid, 65% (v / v) H2O) for 60 minutes, then stained in staining solution (10% acetic acid (v / v), 0.006% (w / v) Coomassie Brilliant Blue R-250, 90% H2O) for 60 minutes. 1_BSH_WT is an unrelated BSH enzyme control that can be distinguished from C. minuta BSH of this disclosure. BSH2_WT is WT C. minuta BSH, i.e., Sequence ID No. 1. [Figure 5B] An exemplary polyacrylamide gel demonstrating the successful expression of the modified BSH enzyme is shown. Staining: Fixed in staining solution (25% (v / v) isopropanol, 10% (v / v) acetic acid, 65% (v / v) H2O) for 60 minutes, then stained in staining solution (10% acetic acid (v / v), 0.006% (w / v) Coomassie Brilliant Blue R-250, 90% H2O) for 60 minutes. 1_BSH_WT is an unrelated BSH enzyme control that can be distinguished from C. minuta BSH of this disclosure. BSH2_WT is WT C. minuta BSH, i.e., Sequence ID No. 1. [Figure 5C] An exemplary polyacrylamide gel demonstrating the successful expression of the modified BSH enzyme is shown. Staining: Fixed in staining solution (25% (v / v) isopropanol, 10% (v / v) acetic acid, 65% (v / v) H2O) for 60 minutes, then stained in staining solution (10% acetic acid (v / v), 0.006% (w / v) Coomassie Brilliant Blue R-250, 90% H2O) for 60 minutes. 1_BSH_WT is an unrelated BSH enzyme control that can be distinguished from C. minuta BSH of this disclosure. BSH2_WT is WT C. minuta BSH, i.e., Sequence ID No. 1. [Figure 5D]An exemplary polyacrylamide gel demonstrating the successful expression of the modified BSH enzyme is shown. Staining: Fixed in staining solution (25% (v / v) isopropanol, 10% (v / v) acetic acid, 65% (v / v) H2O) for 60 minutes, then stained in staining solution (10% acetic acid (v / v), 0.006% (w / v) Coomassie Brilliant Blue R-250, 90% H2O) for 60 minutes. 1_BSH_WT is an unrelated BSH enzyme control that can be distinguished from C. minuta BSH of this disclosure. BSH2_WT is WT C. minuta BSH, i.e., Sequence ID No. 1. [Figure 5E] An exemplary polyacrylamide gel demonstrating the successful expression of the modified BSH enzyme is shown. Staining: Fixed in staining solution (25% (v / v) isopropanol, 10% (v / v) acetic acid, 65% (v / v) H2O) for 60 minutes, then stained in staining solution (10% acetic acid (v / v), 0.006% (w / v) Coomassie Brilliant Blue R-250, 90% H2O) for 60 minutes. 1_BSH_WT is an unrelated BSH enzyme control that can be distinguished from C. minuta BSH of this disclosure. BSH2_WT is WT C. minuta BSH, i.e., Sequence ID No. 1. [Figure 5F] An exemplary polyacrylamide gel demonstrating the successful expression of the modified BSH enzyme is shown. Staining: Fixed in staining solution (25% (v / v) isopropanol, 10% (v / v) acetic acid, 65% (v / v) H2O) for 60 minutes, then stained in staining solution (10% acetic acid (v / v), 0.006% (w / v) Coomassie Brilliant Blue R-250, 90% H2O) for 60 minutes. 1_BSH_WT is an unrelated BSH enzyme control that can be distinguished from C. minuta BSH of this disclosure. BSH2_WT is WT C. minuta BSH, i.e., Sequence ID No. 1.

[0026] [Figure 6A]The chart shows a pie chart illustrating the equilibrium of BA in a healthy state, a NASH state, and a NAFLD state. The data in the chart is based on Puri, P. et al., THE PRESENCE AND SEVERITY OF NONALCOHOLIC STEATOHEPATITIS IS ASSOCIATED WITH SPECIFIC CHANGES IN CIRCULATING BILE ACIDS. Hepatology. 2018 Feb;67(2):534-548, the entire literature of which is incorporated herein by reference. [Figure 6B] The chart shows a pie chart illustrating the equilibrium of BA in a healthy state, a NASH state, and a NAFLD state. The data in the chart is based on Puri, P. et al., THE PRESENCE AND SEVERITY OF NONALCOHOLIC STEATOHEPATITIS IS ASSOCIATED WITH SPECIFIC CHANGES IN CIRCULATING BILE ACIDS. Hepatology. 2018 Feb;67(2):534-548, the entire literature of which is incorporated herein by reference.

[0027] [Figure 7] A pie chart illustrates a novel approach disclosed herein that utilizes a novel enzyme with increased BA deconjugation activity to shift the BA equilibrium from a diseased state back to a healthy state. [Modes for carrying out the invention]

[0028] This disclosure provides modified Christensenella minuta bile acid hydrolase (BSH) enzyme, pharmaceutical compositions comprising the modified BSH enzyme, polynucleotides encoding the modified BSH enzyme, cells (including, but not limited to, bacterial cells) comprising the modified BSH enzyme, cells (including, but not limited to, bacterial cells) comprising the disclosed polynucleotides, and methods of using the modified BSH enzyme, cells (e.g., bacterial cells), and pharmaceutical compositions as probiotics or therapeutic agents for the treatment of human and animal diseases, for improving the health and growth of livestock and companion animals, for agricultural and biofuel applications. The disclosed compositions and methods are intended to shift the bile acid balance to a healthy state while preserving the BA pool.

[0029] Bile salt hydrolases catalyze the hydrolysis of conjugated bile acids to unconjugated bile acids, releasing amino acids such as glycine or taurine. For example, the disclosed modified BSH enzyme has been experimentally shown to hydrolyze the bile acid glycochenodeoxycholic acid (GCDCA) to produce chenodeoxycholic acid and free glycine, and to hydrolyze glycocholic acid to produce cholic acid and glycine. However, the disclosed modified BSH enzyme is thought to hydrolyze further bile acid species.

[0030] Where used herein, “manipulated BSH” and “modified BSH” are interchangeable. Furthermore, in the context of this disclosure, BSH is understood to mean Christensenella minuta BSH unless otherwise specified.

[0031] Modified Christensenella minuta bile salt hydrolase enzyme One aspect of the present disclosure provides a modified Christensenella minuta bile salt hydrolase enzyme. The modified enzyme comprises 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.

[0032] Sequence ID 1 is as follows: MCTAITYYTKDHYFGRNLDLEFSYNETVTVTPKYYPFHFRNGKVLNHHYAMIGMAYIVDDFPLYYDATNEKGLSMAGLNFPDNADYKEVKEGYDNIAPFEFIPWILGQCASVSEARILLEQINLVNLNFSEELPLSPLHWMISDQRDSIVVESTKDGLKVFE NPVGVLTNNPTFDYQMFNLNNYMHLSKEPPANTFAAELELEQYSRGMGAIGLPGDLSSASRFVKAAFTKMNSVSGDSESESISQFFHILGSVEQQRGCVHLGEDKYEITIYSSCCNMDKGIYYYTTYENNQITAVDMYKENLDGNTIISYPLMKEQQINYRNY

[0033] As used herein, “substitution” or “substitutional modification” are used interchangeably and refer to the replacement of one amino acid with another. The disclosed modified enzyme may be at least about 90% identical to SEQ ID NO: 1. The disclosed modified BSH enzyme may be 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 bile salt hydrolase of C. minuta. 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.

[0034] 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 sequence numbers 2-47. The modified enzyme may contain one of sequence numbers 5, 6, 8, 9, 36, 37, and 41-47.

[0035] 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%, and at least about 99% identity with SEQ ID NO: 1, and the modified enzyme may have substitution 157V with respect to SEQ ID NO: 1.

[0036] 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%, and at least about 99% identity with SEQ ID NO: 6, and the modified enzyme has the substitution Y64S with respect to SEQ ID NO: 1.

[0037] 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%, and at least about 99% identity with SEQ ID NO: 1, and the modified enzyme may have substitution S136A with respect to SEQ ID NO: 1.

[0038] 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%, and at least about 99% identity with SEQ ID NO: 1, and the modified enzyme has substitution K189P with respect to SEQ ID NO: 1.

[0039] The modified enzyme may contain sequences 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: 1, and the modified enzyme may have substitutions Y8V, P81A, S206G, and R207Q with respect to SEQ ID NO: 1.

[0040] The modified enzyme may contain sequences 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: 1, and the modified enzyme may have substitutions Y34K, I57V, L127I, and N292S with respect to SEQ ID NO: 1.

[0041] The modified enzyme may contain sequences 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: 41, and the modified enzyme may have substitutions Y56T, I57V, L127I, and N292S with respect to SEQ ID NO: 1.

[0042] 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%, and at least about 99% identity with SEQ ID NO: 1, and the modified enzyme may have substitutions K90E, L127I, N292S, and R323E with respect to SEQ ID NO: 1.

[0043] The modified enzyme may contain sequences that have 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: 43, and the modified enzyme may have substitutions Y34N, H47N, Y64F, Y176K, and A221P with respect to SEQ ID NO: 1.

[0044] The modified enzyme may contain sequences 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: 1, and the modified enzyme may have substitutions Y34N, N46K, H47N, Y64F, Y176K, and A221L with respect to SEQ ID NO: 1.

[0045] The modified enzyme may contain sequences 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: 1, and the modified enzyme may have substitutions Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L with respect to SEQ ID NO: 1.

[0046] The modified enzyme may contain a sequence having at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, and at least approximately 99% identity with any one of SEQ ID NOs. 1 has the substitutions shown in Table 1.

[0047] Modified enzymes may exhibit increased activity compared to wild-type C. minuta BSH at approximately pH 5. Modified enzymes may exhibit increased activity compared to wild-type C. minuta BSH at approximately pH 7. Modified enzymes may exhibit increased activity compared to wild-type C. minuta BSH at approximately pH 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0048] In one aspect of this disclosure, a modified BSH enzyme is provided. The modified BSH enzyme is located at positions 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, S7 4A, 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, S1 53Q, 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, G252D, E255R The amino acid substitutions in 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 may be included.The modified enzyme could 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 sequence number 46.

[0049] Modified BSH enzymes are located at positions 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, L63I, Y64F, Y65K, D66M, G77A, L78V, and N79 relative to SEQ ID NO: 1 S, F80L, D82G, N83I, D85S, K87G, V89K, K90T, Y93K, D94E, F99Y, F101L, W104Y, Q108R, C109A, A110S, S113E, R116L, I117K, Q121N, I 122L, 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, L200I, E201K, Q204R, Y205I, R207A, M209D, A211N, L218K, S220D, A221L , R223K, V225I, K226R, A228T, T230V, K231R, M232L, S234A, S239T, S241D, S243Q, I244L, S245M, G252D, E255R, Q256D, Q257V, R258 P, 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 may contain amino acid substitutions.The modified enzyme could 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 sequence number 47.

[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 any one of the sequence numbers 2–47. The modified enzyme may contain any one of the substitutional mutations disclosed herein; see, for example, Table 1.

[0051] Several C. minuta BSH variants were created and listed below to correspond to the identity of the variants using experimental naming conventions. For example, the variant containing the I57V mutation relative to sequence number 1 is designated as BSH2_3.

[0052] [Table 1]

[0053] Referring here to Figures 3A, 3B, 4A, and 4B, the inventors have demonstrated that several substitutions result in distinguishable properties of the modified BSH enzymes of this disclosure. For example, BSH2_0, BSH2_4, BSH2_6, BSH2_7, BSH2_40, BSH2_42, BSH2_43, BSH2_44, BSH2_45, BSH2_34, and BSH2_35 showed increased GCA hydrolysis 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 showed increased GCDCA hydrolysis compared to wild-type BSH.

[0054] As those skilled in the art will understand, various compartments of the digestive systems of humans and non-human animals have different levels of acidity / alkalinity. For example, intraluminal pH changes rapidly from high acidity in the stomach to approximately pH 6 in the duodenum. pH gradually increases in the small intestine from pH 6 to approximately pH 7.4 in the terminal ileum. pH decreases to 5.7 in the cecum but gradually increases again, reaching pH 6.7 in the rectum. Accordingly, we intend that the disclosed modified BSH enzyme may have different or complementary applications due to its potential differential pH-related activity.

[0055] The disclosed data also demonstrate that certain substitutions resulted in decreased or absent activity in the modified BSH enzyme (e.g., the C2S substitution inactivated the enzyme). See, for example, Figures 3A, 3B, 4A, and 4B.

[0056] The disclosed modified BSH enzyme may be produced in a recombinant system, and for example, the modified BSH enzyme may be expressed by mammalian cells, such as human fetal kidney (HEK) cells, insect cells, fungal cells, such as yeast, and bacterial cells, such as E. coli (Escherichia coli), including but not limited to these.

[0057] In some embodiments, the modified BSH enzymes of this disclosure may include, but are not limited to, one or more 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 labeled or detectable markers; one or more chemical moieties for linking or encapsulation on a solid or semi-solid support, such as beads, carrier molecules, or targeted delivery molecules such as proteins or viruses.

[0058] Polynucleotides In one aspect of the present disclosure, a polynucleotide is provided. In some embodiments, the polynucleotide comprises a sequence encoding a modified Christensenella minuta bile salt hydrolase (BSH) enzyme of the present disclosure.

[0059] A polynucleotide may contain one of sequence numbers 48-79. A polynucleotide may contain one or more regulatory elements. As used herein, “regulatory element” refers to a polynucleotide sequence that modulates the expression of another polynucleotide sequence, such as a promoter or enhancer.

[0060] As used herein, “operatably linked” means a functional linkage between two or more sequences such that activity in one sequence or activity in one sequence affects activity in another sequence. For example, an operatable linkage between a polynucleotide of interest, e.g., a sequence encoding the modified BSH enzyme of this disclosure, and a regulatory element (e.g., a promoter) is a functional linkage that enables the expression of the polynucleotide of interest.

[0061] The promoter may be a constitutive promoter or an inductive promoter. Various promoters are known in the art, for example, SEQ ID NOs: 48-52 or 58-61. See Table 2 for further details.

[0062] The promoter may be inducible depending on environmental conditions.

[0063] The promoter may be responsive to hypoxic or anaerobic conditions.

[0064] [Table 2]

[0065] As used herein, “selection marker” refers to any marker that can be used to select for the expression of a polynucleotide (e.g., antibiotic resistance, nutritional requirements, fluorescence, etc.). Preferred selection markers are known in the art.

[0066] The terms “sequence identity %”, “identity %”, or “identity %” refer to the percentage of amino acid residue matches between at least two amino acid sequences aligned using a standardized algorithm. Methods for amino acid sequence alignment are well known. Some alignment methods consider conserved amino acid substitutions. Such conserved substitutions, described in more detail below, generally preserve charge and hydrophobicity at the substitution site, thereby preserving the structure (and thus the function) of the polypeptide. The amino acid sequence identity % 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 set of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI), and the Basic Local Alignment Search Tool (BLAST) is available on its website from several sources, including NCBI, Bethesda, and Maryland. The BLAST software suite includes various sequence analysis programs, including “blastp,” which are used to align known amino acid sequences with other amino acid sequences from various databases.

[0067] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to polymers of multiple amino acid residues linked to one another by peptide (amide) bonds. These terms refer to proteins, peptides, or polypeptides of any size, structure, or function. Typically, proteins, peptides, or polypeptides are at least three amino acid long. Proteins, peptides, or polypeptides can mean individual proteins or groups of proteins. One or more amino acids in a protein, peptide, or polypeptide may be unnatural and unmodified, or may be added, for example, by the addition of chemically distinctive parts (such as hydrocarbon groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation, functionalization, labeling, or other modifications). Proteins, peptides, or polypeptides may also be single molecules or multimolecular complexes. Proteins, peptides, or polypeptides may be mere fragments of naturally occurring proteins or peptides. Proteins, peptides, or polypeptides may be naturally occurring, recombinant, synthetic, or any combination thereof. The protein may contain different domains, such as a nucleic acid binding domain and a nucleic acid cleavage domain. In some embodiments, the protein includes a proteinaceous moiety, such as an amino acid sequence that constitutes the nucleic acid binding domain.

[0068] The nucleic acids, proteins, and / or other compositions described herein may be purified. As used herein, “purified” means separated from most other compounds or characteristic components, and includes partially or substantially purified. Purity may be indicated by units of weight and may be determined using a variety of analytical techniques, including but not limited to mass spectrometry and HPLC.

[0069] Polypeptide sequence identity may be measured over the entire length of a defined polypeptide sequence, for example, as defined by a specific sequence number, or over a shorter length, for example, over the length of a fragment obtained from a larger defined polypeptide sequence, e.g., over 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 illustrative only, and it is understood that any fragment length for which an identity percentage can be measured may be described herein using any fragment length supported by sequences shown in tables, figures, or sequence listings.

[0070] The disclosed modified BSH enzymes may be delivered to a subject or host by any method including, but not limited to, delivery methods such as biological, static, chemical, nanoparticle, or synthetic methods.

[0071] Method for producing modified Christensenella minuta bile salt hydrolase enzyme One aspect of the present disclosure provides a method for producing the modified BSH enzyme disclosed. In some embodiments, the method for producing the modified BSH enzyme comprises introducing the polynucleotide of the present disclosure into cells, the cells expressing the polynucleotide. The method may further include purifying, concentrating, or isolating the modified BSH enzyme using chromatographic methods, including but not limited to liquid chromatography (LC), high-performance liquid chromatography (HPLC), size exclusion chromatography (SEC), and affinity chromatography, and other methods known in the art.

[0072] Prebiotics In one aspect of the present disclosure, a prebiotic is provided. In some embodiments, the prebiotic comprises one or more of the modified BSH enzymes disclosed. The prebiotic may further comprise one or more additional components for stabilizing or preserving the modified BSH enzymes of the present disclosure.

[0073] As used herein, “prebiotics” refers to indigestible food components that promote the growth of beneficial microorganisms in the digestive tract of animals (e.g., the intestines).

[0074] Prebiotics are thought to modify the target gut microbiome by increasing the amount of unconjugated bile acids. Furthermore, increasing unconjugated bile acids can improve lipid uptake from food in livestock, thereby improving their growth and health.

[0075] As discussed above, the modified BSH enzymes of this disclosure have distinguishable properties and may be selected or combined based on properties to be included in the prebiotic composition, such as activity, selectivity, and activity at a specific pH. The prebiotic may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different modified enzyme types, i.e., modified BSH enzymes with distinguishable substitutions.

[0076] Manipulated bacterial cells In one aspect of this disclosure, engineered bacterial cells are provided. In some embodiments, the bacterial cells comprise a polynucleotide comprising a sequence encoding a modified BSH enzyme disclosed. The engineered bacteria are safe, exhibit good tolerance, and can engraft in a host to improve the host's microbiome, thereby expanding therapeutic benefits.

[0077] The disclosed bacterial cells have been engineered to express a modified BSH enzyme. The engineered bacterial cells are thought to increase the amount of unconjugated bile acids in the subjects, which is associated with a reduced incidence of various human and animal diseases and disorders, including but not limited to non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, metabolic syndrome, and liver cancer.

[0078] The manipulated bacterial cells may be, for example, Christensenella minuta bacterial cells, or one or more bacteria included in the following non-limiting list of exemplary bacterial types, but may be other suitable bacterial cells: Asidaminococcus, Actinomyces, Ackermansia muciniphylla, Alobaculum, Anaerococcus, Anaerostipes, Bacteroides, Bacteroides tetaiotaomicron, Bacteroides vulgatus, Bacteroides tetaiotaomicron VPI-5482, Bacteroides fragilis NCTC-9343, Bacteroides ovatus, Bacteroides uniformis, Bacteroides egertii, other Bacteroides, Bacteroides acidifaciens, Bacteroides coprofil Bacteroides fragilis, Bacteroides ovatus, Bacteroides uniformis, Valnesiellaceae genus, Bifidobacterium adolescentis, Bifidobacterium and others, Bifidobacterium genus, Birophylla genus, Brautia obeum, Brautia producta, Brautia and others, Brautia genus, Burraydia genus, Catenibacterium genus, Chrysenella genus, Citrobacter genus, Clostridaceae genus, Clostridiales other, Clostridiales genus, Clostridium perfringens, Clostridium *Clostridium*, *Chorthella aerofasciens*, *Chorthella*, *Chorthella stercolis*, *Coprococcus catus*, *Coprococcus*, *Coriobacteriaceae*, *Desulfovibrionium*, *Dialistus*, *Drea formisigenerans*, *Drea*, *Drea*, *Aegatella lenta*, *Enterobacteriaceae*, *Enterobacteriaceae*, *Enterococcus*, *Erysipelotrichaseae*, *Eubacterium biforme*, *Eubacterium biforme*, *Eubacterium doricum*, *Eubacterium* Genus, Faecalibacterium prausnitzii, Fusobacterium, Gemeraceae, Haemophilus parainfluenzae, Haemophilus and others, Helicobacter, Helicobacter lachnospiraceae and others, Lachnospiraceae, Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus zeae, Lactobacillus, Lactobacillaceae, Lactococcus, Leuconostocaceae, Megamonas, Megasphere, Metanobrevibacter, Atractylodes japonica, Atractylodes japonica, Musispirium sheldreri,Odolibacter, Osirospira, Parabacteroides distasonis, Parabacteroides, Paraprevotella, Paraprevotellaceae, Parvimonas, Pediococcus, Pediococcus and others, Peptococcus, Peptoniphyllus, Peptostreptococcus anaerobius, Peptostreptococcus and others, Phascorachtobacterium, Prevotella copri, Prevotella, Prevotella stercorea, Prevotellaceae, Proteus, Lyceneraceae, Rosebria fesis, Rosebria, Ruminococcusceae and others, Ruminococcus, Ruminococcus bromii, Ruminococcus gunavas, Ruminococcus, Ruminococcus and others, Ruminococcus turkes, Slacchia, S24-7 spp., SMB53 spp., Streptococcus anginosus, Streptococcus lutesiae, Streptococcus genus, other Streptococcus species, Stellera genus, Tulicibacter genus, UC Braidia, UC Enterobacteria seae, UC Faecalibacterium, UC Parabacteroides, UC Pediococcus, Baribaculum genus, Veillonella genus, Sattara, Tulicibacter, UC Clostridial, UC Erysipelotrichacea, UC Luminococcea, Veillonella parvula, Veillonella genus, Veillonella dispa, s__Tullicibacter_sp001543345:1, and Weissella genus.

[0079] Preferably, the engineered bacterial cells may be selected based on their compatibility with a specific host. For example, in the case of engineered bacterial cells designed to be administered to or provided to humans, bacteria that are normal components of the human microbiome, such as the gut microbiome, such as C. minuta, may be selected. If C. minuta is selected as the bacterial cell, the innate BSH enzyme in the cells may be disrupted or knocked out, leaving only the expression of the modified BSH enzyme.

[0080] A bacterial cell may contain a polynucleotide comprising a sequence encoding one or more of the modified BSH enzymes of this disclosure, which are either present and integrated into the bacterial cell genome, or present and ectopically expressed, i.e., not integrated into the bacterial cell genome. A bacterial cell may contain a plurality of polynucleotides, each polynucleotide comprising a nucleic acid sequence encoding one or more of the modified BSH enzymes of this disclosure.

[0081] The polynucleotides comprising the sequence encoding the modified BSH enzyme of this disclosure may include a “heterogeneous promoter,” i.e., a promoter that is not innate to a particular bacterial cell. For example, the bacterial cell may be a Christensenella minuta cell, and the promoter may be of different biological origin. In some embodiments, the heterogeneous promoter is functionally ligated to the polynucleotide sequence encoding the modified BSH enzyme, and as a result, the engineered cell can express the modified BSH enzyme.

[0082] Probiotics In one aspect of the present 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.

[0083] As used herein, “probiotics” refers to live microorganisms intended to have health benefits when ingested or applied to the body. Probiotics may be used in dietary supplement compositions, as also disclosed herein. Probiotics are also known as live biotherapy products (LBPs).

[0084] For example, probiotic formulations that are ingested or delivered are considered to be publicly known and commonplace in the art.

[0085] As discussed above, the provision of the disclosed modified enzyme, for example in the form of probiotics including engineered bacterial cells expressing the modified enzyme, is considered effective in increasing the amount of unconjugated bile acids in the intestines of subjects, such as human subjects or non-human animal subjects.

[0086] Since the disclosed modified enzymes have potentially different pH values, different properties, e.g., activity, and potentially specificity, the probiotics may comprise two or more types of engineered bacterial cells. For example, the probiotics may comprise two or more types of bacterial cells. The probiotics may further comprise a first type of engineered bacterial cell comprising a specific modified BSH enzyme, and a second type of engineered bacterial cell comprising a second different modified BSH enzyme. Different types of engineered bacterial cells may be selected to occupy different niches in the host (subject) microbiome.

[0087] The disclosed probiotics may be used in several ways, for example, the disclosed probiotics may be provided or administered to a subject.

[0088] The disclosed probiotics may be used in a method for improving livestock yields, which includes providing an effective amount of the disclosed probiotics for improving livestock yields.

[0089] Domesticated animals may include, for example, ruminants, pigs, birds, fish, crustaceans, or mollusks. Ruminants may include, for example, cattle, sheep, goats, deer, buffalo, or camels.

[0090] Animal health

[0091] In livestock, the BSH enzyme plays a crucial role in fat digestion by breaking down bile salts, which are essential for the absorption of dietary fats. This helps improve the nutritional efficiency of animals as well as their overall health and capabilities. Despite differences in lipid metabolism between humans and livestock, BSH remains an important enzyme in gut bacteria. Ruminants such as cattle and sheep have a unique digestive system that includes a specialized stomach space called the rumen. In the rumen, microbial fermentation of plant material occurs, and the resulting fatty acids and lipids can be absorbed in the lower digestive tract after being modified by the gut microbiota. The modified BSH enzyme 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 for energy. Non-ruminant livestock such as pigs and poultry have a simpler digestive system without a rumen. In these animals, the modified BSH enzyme of this disclosure primarily assists in the digestion of dietary fats in the small intestine. These hydrolyze bile salts, making them less effective at emulsifying fats and enabling efficient digestion and absorption of these fats by animals. The fatty acid profile may also be affected. In ruminants, the involvement of microbial fermentation and modified BSH enzymes can lead to alterations in the fatty acid profile of fats absorbed from the digestive tract. This can increase the proportion of volatile fatty acids (e.g., acetates, propionates, butyrates) used for energy and microbial protein synthesis in the ruminants themselves, as well as for milk and meat production.

[0092] In non-ruminant livestock, administration of modified BSH enzymes can contribute to the efficient absorption of dietary fats, leading to broad-spectrum fatty acid uptake into the animal's own tissues, which can affect the fat composition in 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 improved availability and accessibility of animal protein sources for human consumption.

[0093] Modified BSH enzymes contribute to better digestion and absorption of dietary fats in livestock. When animals can efficiently utilize nutrients in their feed, it leads to improved feed conversion efficiency. This means that less feed is needed to produce a given amount of meat, milk, or eggs. As a result, food resources are used more efficiently, and animal protein production becomes more sustainable.

[0094] Livestock that benefit from improved digestion and health through administration of the modified BSH enzyme of this disclosure are likely to exhibit better growth rates, higher milk production, or increased egg production. This increase in productivity can result in greater food production from the same number of animals, thereby contributing to food safety by meeting the requirements for animal protein.

[0095] Healthy livestock are more resistant to disease, reducing the need for antibiotics and other medications. This, in turn, can reduce the risk of antibiotic-resistant pathogens emerging and affecting human health, which is an important aspect of food safety and assurance.

[0096] By improving the efficiency of livestock production, the administration of the modified BSH enzyme of this disclosure may help reduce the environmental footprint of livestock farming. This can result in reduced land and resource use, reduced greenhouse gas emissions, and reduced water consumption per unit of food produced. This is consistent with sustainable agricultural practices and contributes to long-term food security by ensuring the availability of resources for future generations.

[0097] Improving livestock productivity and health can help reduce food loss along 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.

[0098] As described in this section, “administration” of modified BSH enzyme may include the administration of the enzyme as part of a therapeutic / nutritional composition, and / or the administration of cells expressing the enzyme as part of a therapeutic / nutritional composition.

[0099] Aquaculture

[0100] Despite the vastly different methods of lipid metabolism (fish have a much simpler digestive system than humans), modern aquaculture techniques involve feeds containing high levels of fat and lipids. The modified BSH enzymes of this disclosure can be added to the diet, for example, as prebiotics or probiotics, to enhance the ability to digest and absorb these nutrients. This can result in better growth rates and improved feed conversion efficiency, which is crucial for maximizing the productivity of aquaculture operations. By improving fat and lipid digestion, the administration or provision of the modified BSH enzymes of this disclosure can help fish and other aquatic organisms better utilize the nutrients present in the feed. This results in more efficient conversion of nutrients to body weight, reduces the amount of waste generated, and potentially improves water quality in aquaculture systems.

[0101] The modified BSH enzyme of this disclosure may enable the use of alternative feed components in aquaculture feeds. Some feed components, such as plant proteins and oils, may contain antitrophic factors, such as phyticates, that bind to minerals and reduce their bioavailability. Providing the administration of the modified BSH enzyme of this disclosure can help break down these compounds, enabling the incorporation of a wider range of feed components while maintaining nutritional value.

[0102] Improving nutrient utilization and growth efficiency can lead to a reduction in the environmental impact of aquaculture operations. When fish and other aquatic organisms grow more efficiently and produce less waste, there is less nutrient runoff and pollution of the surrounding aquatic ecosystem.

[0103] The provision or administration of the modified BSH enzyme described herein is thought to have a positive effect on the health of aquaculture species. Bile salts can have antibacterial properties, and by degrading them, the modified BSH enzyme can potentially reduce the carrier rate of harmful pathogens in the intestines. This may help reduce the incidence of disease in aquaculture populations.

[0104] Food security

[0105] The provision or administration of modified BSH enzymes to livestock can contribute to food security by improving the efficiency, sustainability, and resilience of livestock farming, ultimately leading to improved availability and accessibility of animal protein sources for human consumption. Improved nutrient utilization: Modified BSH enzymes can contribute to better digestion and absorption of dietary fats in livestock. When animals can efficiently utilize nutrients in their feed, it leads to improved feed conversion efficiency. This means that less feed is needed to produce a given amount of meat, milk, or eggs. As a result, food resources are used more efficiently, and animal protein production becomes more sustainable.

[0106] Livestock that benefit from improved digestion and health through administration of the modified BSH enzyme of this disclosure are likely to exhibit better growth rates, higher milk production, or increased egg production. This increase in productivity can lead to greater food production from the same number of animals, thereby contributing to food security by meeting the demand for animal protein. Improved animal health: Healthy livestock are more resistant to disease and reduce the need for antibiotics and other pharmaceuticals. This, in turn, can reduce the risk of antibiotic-resistant pathogens emerging and affecting human health, which is an important aspect of food safety and security.

[0107] By improving the efficiency of livestock production, providing the modified BSH enzyme administration of this disclosure may help reduce the environmental footprint of livestock farming. This can result in reduced land and resource use, reduced greenhouse gas emissions, and reduced water consumption per unit of food produced. This is consistent with sustainable agricultural practices and contributes to long-term food security by ensuring the availability of resources for future generations. Enhancing livestock productivity and health may help reduce food loss along 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.

[0108] Bioremediation

[0109] The modified BSH enzymes of this disclosure can be used in bioremediation processes to help decompose and remove contaminants in water and soil. Some contaminants, such as certain hydrophobic organic compounds, may be difficult to decompose in nature. The modified BSH enzymes of this disclosure may help enhance the decomposition of these compounds by altering their interactions with bile salts and microbial communities.

[0110] kit In one aspect of the present disclosure, a kit is provided. The kit may include: nucleic acids encoding one or more of the modified BSH enzymes of the present disclosure; optionally, cells for transformation or transfection with nucleic acids; modified BSH enzymes; bacterial cells capable of expressing one or more of the modified BSH enzymes; pharmaceutical compositions comprising cells expressing one or more of the BSH enzymes and / or one or more of the modified BSH enzymes; medical foods comprising cells expressing one or more of the BSH enzymes and / or one or more of the modified BSH enzymes; and optionally, instructions for carrying out any of the methods disclosed.

[0111] Pharmaceutical composition In one aspect of the present disclosure, a pharmaceutical composition is provided. In some embodiments, the pharmaceutical composition comprises engineered bacterial cells of the present disclosure that can express one or more modified BSH enzymes. In some embodiments, the pharmaceutical composition comprises one or more of the modified BSH enzymes of the present disclosure.

[0112] The pharmaceutical composition may further contain pharmaceutically acceptable carriers or excipients, and the formulations thereof are commonplace and readily understood by those skilled in the art.

[0113] method In one aspect of this disclosure, a method is provided. In some embodiments, the method comprises administering a pharmaceutical composition comprising one or more of the modified BSH enzymes described herein and / or one or more cells expressing the modified BSH enzymes of this disclosure to a subject requiring it.

[0114] As used herein, “required subjects” or “subjects” may refer to vertebrates, such as livestock (e.g., cattle, chickens, goats, sheep, llamas, alpacas, camels, horses, donkeys), fish, crustaceans, mollusks, 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 embodiments, subjects are humans. In some embodiments, subjects are cats. Required subjects may refer to humans suffering from diseases or disorders associated with NASH, NAFLD, Alzheimer's disease, Crohn's disease, cholestatic disease, or metabolic syndrome.

[0115] The relative abundance of the bacterium *C. minuta* in the human gut is positively correlated with the phenotype of lean hosts associated with a low BMI index. See, for example, Ang, W. et al. A Keystone Gut Bacterium *Christensenella minuta* - A Potential 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; these are incorporated herein by reference in their entirety. Furthermore, modified *C. minuta* bacteria have been studied (NCT04663139).

[0116] Fatty liver disease (also known as hepatic lipidosis) in cats typically results from prolonged loss of appetite or starvation. When a cat ceases feeding for an extended period, its body mobilizes fat stores to meet its energy requirements, leading to an excessive accumulation of fat in the liver. This accumulation impairs liver function, impairing its ability to perform essential functions such as detoxification and metabolism. Without prompt intervention, fatty liver disease progresses rapidly, potentially leading to severe liver dysfunction, jaundice, and potentially liver failure. Treatment often involves aggressive nutritional support, including forced feeding or the placement of a feeding tube to ensure adequate calorie intake. Veterinary care may also include supportive measures such as fluid therapy, vitamin supplementation, and drug therapy to support liver function. Despite treatment efforts, the prognosis for cats with hepatic lipidosis can vary depending on the severity of the disease and the presence of underlying conditions. Many cats can recover with timely intervention, but a significant percentage may succumb to complications associated with advanced liver disease. Prevention, early detection, and intervention are crucial to improving outcomes and minimizing mortality risk in cats with fatty liver disease. Modified enzymes disclosed as pharmaceutical compositions or probiotics in food are thought to be able to treat signs or symptoms of feline hepatic lipidosis.

[0117] In some embodiments, the method is a method for treating non-alcoholic fatty liver disease (NAFLD) in a subject requiring treatment for NAFLD, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of the present disclosure in order to treat the NAFLD.

[0118] As used herein, “therapeutic dose” or “effective dose” refers to the amount or dosage of a pharmaceutical composition that, in a single or multiple administration to a subject, provides a desired effect in a subject under diagnosis or treatment. For example, a therapeutic dose of a pharmaceutical composition of this disclosure may include an amount effective in improving 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 disease, such as coronary artery disease, peripheral artery disease, carotid artery disease, heart failure, and stroke.

[0119] C. minuta has been reported to be absent from the microbiome of patients with Crohn's disease (CD), and has been demonstrated 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 Christensenella minuta as a future biotherapy for Crohn's disease. Sci Rep. 2022;12:6017, which is incorporated herein by reference.

[0120] The disclosed pharmaceutical compositions may 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.

[0121] Cholestatic disorders refer to a group of conditions characterized by impaired bile flow from 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 disorders, bile acid dysfunction results from the disruption of the normal bile flow process. This dysfunction can lead to a variety of symptoms, including jaundice, itching, fatigue, and, in severe cases, liver damage.

[0122] Bile salt hydrolase (BSH) is an enzyme that plays a crucial role in the metabolism of bile acids. It catalyzes the hydrolysis of bile salts into bile acids and amino acids. In the context of cholestatic diseases, where bile acid accumulation is present, BSH may serve as a therapeutic target. By enhancing BSH activity, excess bile salts can be broken down into bile acids, thereby reducing their toxic effects on hepatocytes and improving bile flow. This mechanism helps alleviate symptoms associated with cholestatic diseases and can further prevent further liver damage. Therefore, the use of novel BSH with higher activity as a therapeutic intervention is promising for the treatment of cholestatic diseases by restoring bile acid homeostasis and promoting liver health. For example, see Li, T. and Apte, U. Bile acid metabolism and signaling in cholestasis, inflammation and cancer Adv Pharmacol. 2015;74:263-302, and Zheng, J. et al. Bile acid-mediated signaling in cholestic liver diseases Cell&Bioscience volume 13, Article number:77(2023), each of which is incorporated herein by reference in its entirety.

[0123] Examples of cholestasis disorders may be treated using the disclosed pharmaceutical compositions, probiotics, prebiotics, and methods:

[0124] 1. Primary biliary cholangitis (PBC): This is an autoimmune disease that primarily affects adults, especially middle-aged women. It results in inflammation and destruction of the small bile ducts within the liver.

[0125] 2. Primary sclerosing cholangitis (PSC): Another autoimmune disorder, 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.

[0126] 3. Biliary atresia: This is a rare condition that affects newborns and infants. It involves complete or partial obstruction of the bile ducts outside or inside the liver, leading to bile buildup, liver damage, and ultimately cirrhosis if left untreated.

[0127] 4. Alagille syndrome: This genetic disorder affects multiple organs, including the liver. Alagille syndrome is characterized by bile duct abnormalities that lead to cholestasis and liver damage. It can be present in infancy or childhood.

[0128] 5. Progressive Familial Intrahepatic Cholestasis (PFIC): PFIC includes a group of rare genetic disorders that result in bile formation disorders and cholestasis. It typically manifests in infancy or early childhood and can lead to progressive liver damage.

[0129] 6. Benign recurrent intrahepatic cholestasis (BRIC): BRIC is another rare genetic disorder characterized by intermittent episodes of cholestasis and jaundice. It is usually present in late childhood or adulthood and is generally less severe than PFIC.

[0130] As used herein, “treat” and its grammatical variations mean reducing or preventing at least one sign or symptom of a disease or disorder. For example, treatment of NAFLD may include reducing or preventing weakness, loss of appetite, nausea, yellowing of the skin and eyes (jaundice), itching, fluid accumulation and swelling in the legs and abdomen, mental confusion, or gastrointestinal (GI) bleeding. With respect to HCC, treating may mean reducing abdominal discomfort or bloating, jaundice, gastrointestinal bleeding, nausea or vomiting, persistent itching, or fever. With respect to obesity, treating may mean reducing the percentage / composition of body fat. With respect to hyperlipidemia and hypercholesterolemia, treating may mean normalizing the blood lipid or cholesterol profile, respectively. With respect to Alzheimer's disease (AD), treating may mean reducing confusion, aphasia, or other symptoms associated with AD. With respect to Crohn's disease, treating may mean reducing the frequency of bowel movements, reducing pain associated with bowel movements, or gaining weight. In relation to cardiovascular disease, treatment may refer to reducing paralysis, confusion, difficulty speaking or understanding speech, difficulty seeing with one or both eyes, difficulty walking, dizziness, and / or loss of balance or coordination on one side of the face, arms, or legs, particularly on one side of the body.

[0131] In some embodiments, the method is a method for treating non-alcoholic steatohepatitis (NASH) in a subject requiring treatment for NASH, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of the present disclosure to the subject to treat NASH.

[0132] In some embodiments, the method is a method for treating liver cancer in a subject requiring treatment for liver cancer, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present disclosure to the subject to treat the liver cancer.

[0133] In some embodiments, the method is a method for treating Alzheimer's disease in a subject requiring treatment for Alzheimer's disease, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present disclosure to the subject to treat Alzheimer's disease.

[0134] Liver cancer

[0135] The disclosed engineered BSH enzyme has potential as a therapeutic target for liver cancer due to its effects on bile acid metabolism and the gut-liver axis. It can be engineered to disrupt carcinogenic processes and promote anti-cancer mechanisms in the liver. The balance between conjugated and unconjugated bile acids is important in liver cancer; excess conjugated bile acids may promote inflammation and carcinogenesis, while high levels of conjugated bile acids may lead to hepatocyte damage and contribute to carcinogenesis. The disclosed enzyme promotes an increase in unconjugated bile acids, which is a therapeutic effect for treating and preventing liver cancer.

[0136] Alzheimer's disease

[0137] The role of lipid metabolism in Alzheimer's disease is an active area of ​​research, and there is evidence suggesting that dysregulation of lipid metabolism may play a significant role in the onset and progression of the disease. Alzheimer's disease is a complex neurodegenerative disorder characterized by the accumulation of amyloid plaques and tau entanglements in the brain, as well as neuronal cell death and cognitive decline. Cholesterol is a type of lipid essential for brain function, as it is a major component of cell membranes and myelin, the protective sheath around nerve fibers. Abnormalities in cholesterol metabolism, such as high levels of LDL ("bad") cholesterol or low levels of HDL ("good") cholesterol, are associated with an increased risk of Alzheimer's disease. Disruption of cholesterol homeostasis is thought to lead to the accumulation of amyloid-beta plaques, a characteristic feature of Alzheimer's disease.

[0138] Lipid peroxidation, or oxidative degradation of lipids, can lead to the generation 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 disrupt nerve function. Lipid rafts are specialized regions of cell membranes rich in cholesterol and certain lipids. They play a role in the processing of amyloid precursor protein (APP), which is cleaved to form amyloid-beta, a peptide that accumulates in Alzheimer's disease. Changes in lipid rafts can affect APP cleavage and lead to increased amyloid-beta production.

[0139] APOE is a protein involved in lipid transport in the brain. APOE has different isoforms, and the APOEε4 allele is a major genetic risk factor for late-onset Alzheimer's disease. This allele is associated with alterations in lipid metabolism in the brain and may affect the clearance of amyloid-beta from the brain. The lipid composition in the brain can affect membrane integrity and fluidity, which in turn affects neuronal function. Changes in the levels of certain lipids (e.g., sphingolipids and phospholipids) have been observed in the brains of individuals with Alzheimer's disease.

[0140] In some embodiments, the method is a method for treating a metabolic syndrome-related disease or disorder in a subject requiring treatment for a metabolic syndrome-related disease or disorder, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present disclosure to the subject to treat the metabolic syndrome-related disease or disorder. In some embodiments, the metabolic syndrome-related disease or disorder 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.

[0141] As used herein, “metabolic syndrome-related disease or disorder” refers to any disease, disorder, 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.

[0142] The disclosed compositions and methods may be used to treat signs or symptoms of diabetes, such as type 2 diabetes, including, for example, lowering fasting blood glucose.

[0143] Bioremediation

[0144] BSH enzymes can be used in bioremediation processes to help decompose and remove contaminants in water and soil. Some contaminants, such as certain hydrophobic organic compounds, may be difficult to decompose in nature. The modified BSH enzymes of this disclosure may help enhance the decomposition of these compounds by altering their interactions with bile salts and microbial communities.

[0145] food In one aspect of the present disclosure, a food product is provided. In some embodiments, the food product comprises lipids isolated from an organism by further comprising the methods of the present disclosure, for example, contacting an organism with a modified enzyme of the present disclosure, and isolating, purifying, or extracting lipids from the organism.

[0146] Food can take any form, such as meat substitutes or food additives.

[0147] Medical Foods In one aspect of the present disclosure, a medical food is provided. In some embodiments, the medical food comprises a modified BSH enzyme, prebiotics, bacterial cells, or probiotics as described in the present disclosure.

[0148] As used herein, “medical food” means a food that is formulated to be taken or administered enterally under the supervision of a physician and is intended for the specific dietary management of a disease or condition in which characteristic nutritional requirements have been established by medical evaluation, based on recognized scientific principles.

[0149] Medical foods may be administered in any form, including but not limited to powders, pills, yogurts, capsules, tablets, and gels.

[0150] definition The subject matter disclosed may be further described using the following definitions and terms. The definitions and terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit them.

[0151] As used herein and in the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly indicates otherwise. For example, the term “substituent” should be interpreted as “one or more substituents” unless the context clearly indicates otherwise.

[0152] As used herein, “about,” “approximately,” “substantially,” and “significantly” are understood by those skilled in the art and vary to some extent depending on the context in which they are used. Where there is a use of a term that is not clear to those skilled in the art from the context in which it is used, “about” and “approximately” mean up to plus or minus 10% of a particular term, and “substantially” and “significantly” mean more than plus or minus 10% of a particular term.

[0153] 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” transitional terms that allow for the inclusion of additional components in the components described in the claims. The terms “consist” and “consisting of” should be interpreted as “closed” transitional terms that do not allow for the inclusion of additional components other than those described in the claims. The term “essentially consisting of” should be interpreted as partially closed, allowing for the inclusion of only additional components that do not fundamentally alter the nature of the claimed subject matter.

[0154] The phrase "etc." should be interpreted as "for example, including." Furthermore, the use of any and all illustrative language (for example, "etc.", but not limited to) is merely intended to better illustrate the invention and does not impose any limitation on the scope of the invention unless otherwise claimed.

[0155] Furthermore, where conventions similar to “at least one of A, B, and C” are used, such constructions are generally intended to be understood by those skilled in the art (for example, “a system having at least one of A, B, and C” includes, but is not limited to, systems having A only, B only, C only, A and B together, A and C together, B and C together, and / or systems having A, B, and C together). Furthermore, it will be understood by those skilled in the art that virtually any disjunct word and / or disjunct phrase presenting two or more alternative terms should be understood as intending to include the possibility of including one of the terms, either of the terms, or both of the terms, whether in a description or a diagram. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B”.

[0156] All terms such as "up to / maximum," "at least," "greater than," and "less than" include the listed numbers and refer to ranges that can later be divided into ranges and subranges. A range includes individual elements. Therefore, for example, a group having 1 to 3 elements refers to a group having 1, 2, or 3 elements. Similarly, a group having 6 elements refers to a group having 1, 2, 3, 4, 5, or 6 elements, and so on.

[0157] The verb "may" refers to a preferred use or selection of one or more options or choices among several described embodiments or features contained herein. If no options or choices are disclosed with respect to a particular embodiment or feature contained herein, the verb "may" refers to an affirmative action relating to a method or method and manner of making or using a described embodiment or feature contained herein, or a final decision to use a particular technique relating to a described embodiment or feature contained herein. In this latter context, the verb "may" has the same meaning and connotation as the auxiliary verb "can". Exemplary Embodiments 1. Recombinant bacterial cells possessing heterologous BSH genes derived from the genome of Christensenella minuta. 2. Recombinant bacterial cells according to Embodiment 1, wherein the expression of the BSH gene is controlled by a heterologous promoter. 3. Manipulated bacteria from Embodiments 1 and 2, wherein the promoter is an inductive promoter. 4. Manipulated bacteria from Embodiments 1 and 2, wherein the promoter is inducible by environmental conditions including conditions specific to the mammalian gut. 5. Operated bacteria from Embodiments 1-4, wherein the promoter is responsive to hypoxic or anaerobic conditions. 6. Manipulated bacteria from Embodiments 1-2, wherein the promoter is a constitutively active promoter. 7. Manipulated bacteria from embodiments 1 to 6, further comprising heterologous gene sequences encoding one or more BSH genes, including DSM33407. 8. Manipulated bacterial cells from Embodiments 1-7, wherein the BSH gene is directly or indirectly linked to an inducible promoter. 9. Manipulated bacteria from Embodiments 1-7, wherein the promoter is inducible by environmental factors, including those present in the mammalian gut. 10. Operated bacteria from Embodiments 1-7, wherein the promoter is inducible under hypoxic or anaerobic conditions. 11. Manipulated bacteria from embodiments 1-7, wherein the promoter is a constitutive promoter. 12. Modified bacteria from Embodiments 1-7, comprising variants of the gene further modified to obtain improvements in enzyme function, protein stability, broader substrate specificity, or any combination thereof. 13. Engineered bacteria from Embodiments 1-7, wherein the bacteria are engineered to contain a combination of at least one BSH gene originally derived from Christensenella minuta and a second BSH allele derived from another bacterium commonly found in the mammalian gut microbiota. These microbiota-associated bacterial strains include, but are not limited to, the genera Asidaminococcus, Actinomyces, Ackermansia, Ackermansia muciniphylla, Allobaculum, Anaerococcus, Anaerostipes, Bacteroides, Bacteroides others, Bacteroides acidifaciens, Bacteroides coprofilus, Bacteroides fragilis, Bacteroides ovatus, Bacteroides uniformis, Valnesiellaceae, Bifidobacteria Thelium adolescentis, Bifidobacterium and others, Bifidobacterium genus, Bilophila genus, Brautia obeum, Brautia producta, Brautia and others, Brautia genus, Burraydia genus, Catenibacterium genus, Chrysenella genus, Christensenella minuta genus, Citrobacter genus, Clostridiaceae genus, Clostridiales and others, Clostridiales genus, Clostridium perfringens, Clostridium genus, Clostridium and others, Co Lincella aerofasiens, Chorincella genus, Chorincella stercolis, Coprococcus catus, Coprococcus genus, Coriobacterium seae, Desulfovibrion genus, Dialist genus, Dorea formisigenerans, Dorea genus, Dorea others, Aegatacera lenta, Enterobacterium seae others, Enterobacterium seae genus, Enterococcus genus, Erysipelotrichaceae genus, Eubacterium biforme, Eubacterium biforme, Eubacterium Mu Dorikami, Eubacterium, Faecalibacterium prausnitzii, Fusobacterium, Gemeraceae, Haemophilus parainfluenzae, Haemophilus and others, Helicobacter, Helicobacter lachnospiraceae and others, Lachnospiraceae, Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus zeae, Lactobacillus, Lactobacillaceae, Lactococcus, Leuconostocaceae, Megamonas, Megasphere,Metanobrevibacter, Atractylodes japonica, Atractylodes, Musispirium shelderii, Odolibacter, Osirospira, Parabacteroides distasonis, Parabacteroides, Paraprevotella, Paraprevotellaceae, Parvimonas, Pediococcus, Pediococcus and others, Peptococcus, Peptoniphyllus, Peptostreptococcus anaerobius, Peptostreptococcus Leptococcus and others, Phascolarctobacterium, Prevotella copri, Prevotella, Prevotella stercorea, Prevotella family, Proteus, Lycenera family, Rosebria fesis, Rosebria, Ruminococcus family and others, Ruminococcus, Ruminococcus bromii, Ruminococcus gunavas, Ruminococcus, Ruminococcus and others, Ruminococcus turkes, Slacchia, S24-7 Examples include spp., SMB53 spp., Streptococcus anginosus, Streptococcus lutesiae, Streptococcus genus, other Streptococcus species, Stellera genus, Tulicibacter genus, UC Braidia, UC Enterobacteria seae, UC Faecalibacterium, UC Parabacteroides, UC Pediococcus, Baribaculum genus, Veillonella genus, Sattara, Tulicibacter, UC Clostridial, UC Erysipelotrichacea, UC Luminococcea, Veillonella parvula, Veillonella genus, Veillonella dispa, and Weissella genus. 14. Modified bacterial cells from Embodiments 1-13, wherein the BSH gene is derived from the Christinsinella minuta gene and further modified using synthetic biotools to improve the protein half-life, enzymatic activity per unit of protein, bile salt substrate specificity, or any combination of the three features listed herein. 15. Manipulated bacterial cells from Embodiments 1-14, wherein the BSH gene is directly or indirectly linked to an inducible promoter. 16. Manipulated bacteria from Embodiments 1-14, wherein the promoter is inducible by environmental factors, including those present in the intestines of mammals (human and / or non-humans). 17. Manipulated bacteria from Embodiments 1-14, wherein the promoter is inducible under hypoxic or anaerobic conditions. 18. Manipulated bacteria from embodiments 1 to 14, wherein the promoter is a constitutive promoter. 19. Modified bacteria from Embodiments 1 to 14, comprising variants of the gene further modified to obtain improvements in enzyme function, protein stability, broader substrate specificity, or any combination thereof. 20. Modified bacteria from embodiments 1-19, wherein the heterologous BSH-containing gene is originally derived from Christensenella minuta. 21. Engineered bacteria from Embodiments 1-20, wherein the BSH gene is located in a plasmid within the bacterial cell. 22. Manipulated bacteria from Embodiments 1-20, in which the BSH gene is incorporated into the chromosome of the bacterial cell. 23. Manipulated bacterial cells from Embodiments 1-22, which are members of the genera Bacteroides, Bifidobacterium, Clostridium, Escherichia coli, Lactobacillus, Lactococcus, or Turicibacter. 24. A pharmaceutical composition comprising manipulated bacterial cells as described in any one of Embodiments 1 to 23 and a pharmaceutically acceptable carrier. 25. A method for treating a disease related to bile salt dysregulation in a subject requiring treatment for such a disease, comprising administering the pharmaceutical composition described in Embodiment 24 to the subject. 26. A method for reducing the level of bile salts in a target intestine, comprising administering the pharmaceutical composition described in Embodiment 24 to a target, thereby reducing the level of bile salts in the target intestine. 27. A method for reducing conjugated bile salts, unconjugated bile salts, or both conjugated and unconjugated bile salts in a subject, comprising administering the pharmaceutical composition described in 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 intestines of the subject. 28. The method according to Embodiment 25, wherein the disorder related to bile salt dysregulation is a metabolic disorder or hepatic steatosis. 29. The method according to Embodiment 25, wherein the disorder related to bile salt dysregulation is a cardiovascular disease. 30. A method for improving clinical signs or symptoms in a mammal exhibiting NAFLD, comprising administering bacterial cells containing the BSH gene derived from the Christensenella minuta DSM33407 strain, or a functional equivalent of the BSH gene that shares at least 90% identity with the Christensenella minuta DSM33407 strain. 31. The method according to Embodiment 30, wherein bacterial cells express the BSH gene upon introduction into the intestine of a mammal. 32. The method according to any one of Embodiments 25 to 31, wherein bacterial cells are administered as part of a formulation suitable for oral delivery. 33. The method according to any one of Embodiments 1 to 32, wherein the subject is a mammal. 34. The method according to Embodiment 33, wherein the subject is a human. 35. The method according to Embodiment 33, wherein the subject is a dog, cat, horse, ferret, guinea pig, or hamster. 36. The method according to Embodiment 33, wherein the subject is a cattle, goat, sheep, deer, yak, pig, donkey, reindeer, llama, alpaca, camel, or rabbit. 37. The method according to Embodiment 30, wherein the bacterial cells are engineered Christensenella minuta. 38. A method for producing an improved animal product, comprising administering the pharmaceutical composition described in Embodiment 24 to livestock and recovering livestock or products from livestock to produce an improved animal product. 39. The method according to any one of Embodiments 25 to 38, wherein the administration includes intravenous, intramuscular, subcutaneous, rectal, vaginal, or oral administration. 40. The method according to Embodiment 39, wherein the administration includes oral or rectal administration. 41. The method according to Embodiment 40, wherein the administration includes oral administration. [Examples]

[0158] The following embodiments are illustrative and should not be construed as limiting the scope of the claimed subject matter.

[0159] Example 1 - BSH expression Introduction

[0160] the purpose

[0161] The purpose of this protocol is to describe the complete experimental setup, from E. coli culture to the detection of the product after the enzymatic reaction of the cell lysate with the substrate.

[0162] Background information

[0163] In a previous experiment, OD 600 It was confirmed that induction with 100 μM IPTG at approximately 1.0 and subsequent expression at 37°C for 20 hours resulted in BSH expression.

[0164] material

[0165] chemicals

[0166] Isopropyl β-d-1-thiogalactopyranoside (CAS: 367-93-1)

[0167] [Table 3]

[0168] Consumables: Microtiter plates, 2.2 mL square V-bottom deep well plates

[0169] Experimental equipment: plate shaker, centrifuge, spectrophotometer

[0170] method

[0171] Day 1 (preculture)

[0172] Microtiter plates were prepared in 145 μL of LB medium containing 50 μg / mL kanamycin. The variant culture (BSH variant in E. coli C41 pLys) was thawed. After resuspending, 5 μL of the variant was inoculated into each well. A 150 μL MTP was sealed with a lid. The MTP was incubated at 37°C for 16 hours with shaking at 900 rpm.

[0173] 2nd day (main culture)

[0174] To assess cell growth, 5 μL of pre-culture was diluted 1:40 in 195 μL of LB medium. Absorbance at 600 nm was measured. The pre-culture was then diluted in LB medium for the main culture. 600 The solution was diluted to approximately 0.05. 495 μL of LB containing 50 μg / mL kanamycin was added to each well of a deep-well plate. 5 μL of pre-culture was added to the medium. The inoculated medium was then used to determine if the bacterial culture was OD (Oxygen-Derived). 600 Grow at 37°C with shaking at 900 rpm and 80% humidity until the OD reaches approximately 1.0, thereby allowing the OD to develop during culture. 600 OD was measured. 600 After the expression level reached approximately 1.0, the cells were induced to a final concentration of 100 μM IPTG with 5 μL of 10 mM IPTG. After induction, the DWP was sealed in gas-permeable foil. Expression was performed at 37°C for 20 hours under shaking at 900 rpm and 80% humidity.

[0175] collect

[0176] After 20 hours of expression, cells were harvested by centrifugation at 4,000 g for 10 minutes at 4°C. The supernatant was then discarded. The cell pellet was frozen at -20°C for at least 1 hour.

[0177] Example 2 - Taurine Assay Introduction

[0178] the purpose

[0179] The objective of this preview experiment is to determine the taurine concentration after a BSH assay on TCA. The taurine concentration indicates the activity of BSH. See, for example, Figure 2. The following protocol may be performed to detect the activity of the disclosed BSH enzyme, for example, when hydrolyzing taurocholic acid (TCA) to obtain taurine and cholic acid.

[0180] Background information

[0181] Use the sample from Example 1.

[0182] material

[0183] chemicals

[0184] Taurine assay kit (MET-5071, cell biolabs)

[0185] [Table 4]

[0186] Consumables: Transparent MTP F-bottom.

[0187] Experimental equipment: Plate reader, plate shaker

[0188] method

[0189] Detailed protocol

[0190] Preparation of a taurine assay

[0191] 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 the standard material in two batches at concentrations of 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 manual (see Table 3).

[0192] [Table 5]

[0193] Sample preparation

[0194] On the one hand, transfer 50 μL of deproteinized sample from the BSH assay at pH 7 to the MTP. On the other hand, transfer 33 μL of deproteinized sample from the BSH assay at pH 5 to the MTP and add 17 μL of 200 mM TRIS / HCl buffer, pH 9, to these samples. To reach approximately pH 7, it is necessary to add pH 9 buffer to the pH 5 sample. All samples are processed in pairs. Determine the sample background by directly measuring one copy with a taurine kit, while quenching the other copy (see next section - Taurine Assay).

[0195] Taurine assay

[0196] Transfer 50 μL of the standard substance to the MTP. Start the reaction with 50 μL of the reaction mixture. Incubate at 25°C for 30 minutes with shaking. Stop the reaction with 50 μL of stop solution and 50 μL of chromogenic solution. To detect the sample background, quench the reaction with 50 μL of quench solution and 50 μL of chromogenic solution, and incubate at 25°C for 3 minutes with shaking. Detect the absorbance (415 nm) using a plate reader. Subtract the absorption of the sample background (=after quenching) from the absorption of the sample stopped with the stop solution.

[0197] Example 3 - Glycine assay Introduction

[0198] Background information

[0199] The sample from the BSH assay (Example 1) was thawed. The glycine standard and glycine assay buffer from the glycine assay kit were thawed at room temperature. The remaining components were thawed on ice. Standards were prepared in double batches at concentrations of 0 μM, 1.56 μM, 3.13 μM, 6.25 μM, 12.5 μM, 25.0 μM, 50.0 μM, and 100.0 μM. The reaction mixture was prepared according to the manual.

[0200] material

[0201] chemicals

[0202] Glycine assay kit (ab211100, abcam)

[0203] [Table 6]

[0204] Consumables: Black MTP; Laboratory equipment: Plate reader, plate shaker.

[0205] method

[0206] The samples were thawed. The components of the glycine assay kit were thawed at room temperature with the GLY assay buffer, and the remaining components of the kit were placed on ice. A 1 mM standard was prepared by diluting 5 μL of 100 mM GLY standard with 495 μL of ddH2O. A 50 μM standard was prepared by diluting 50 μL of 1 mM GLY standard with 950 μL of ddH2O. Glycine was used as a standard in concentrations of 0 μM, 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM. For each reaction, the reaction mixture was prepared according to the manual (see Table 5).

[0207] [Table 7]

[0208] Sample preparation

[0209] The pH 7 sample was diluted 1:3 in assay buffer. The pH 5 sample was diluted 2:3 in pH 9 buffer, and then further diluted 1:2 in assay buffer.

[0210] Glycine assay

[0211] 50 μL of the standard substance was transferred to a black MTP. 50 μL of a 1:3 diluted sample was transferred to a black MTP. The reaction was initiated with 50 μL of the reaction mixture. Protected from light, the mixture was incubated at 37°C for 30–60 minutes with shaking at 900 rpm. Fluorescence was measured after 60 minutes using an Ex / Em 560 / 587.

[0212] Example 4 - Treatment of non-alcoholic fatty liver disease In one example, a subject suffering from non-alcoholic fatty liver disease (NAFLD) is administered a therapeutically effective dose of a disclosed composition, such as a pharmaceutical composition, prebiotics, or probiotics. The composition may be suitably administered by any route indicated by the subject's specific treatment needs, such as orally or rectally. Signs and symptoms of NAFLD may be alleviated by the administration of the composition. Treatment may be administered daily, every other day, every three days, or on a schedule determined by the patient's progression, as determined by the physician's decision. The subject is expected to undergo improvements in weight, appetite, or nausea, vomiting, abdominal pain, or itching, or other metrics related to the alleviation of signs or symptoms of NAFLD, compared to an untreated subject. Methods for measuring the alleviation of signs and symptoms of NAFLD are known in the art.

[0213] Example 5 - Treatment of non-alcoholic steatohepatitis In one example, a subject suffering from non-alcoholic steatohepatitis (NASH) is administered a therapeutically effective dose of a disclosed composition, such as a pharmaceutical composition, prebiotics, or probiotics. The composition may be suitably administered by any route indicated by the subject's specific treatment needs, such as orally or rectally. The signs and symptoms of NASH may be alleviated by the administration of the composition. Treatment may be administered daily, every other day, every three days, or on a schedule determined by the patient's progression, as determined by the physician's decision. The subject is expected to undergo improvements in weight, appetite, or nausea, vomiting, abdominal pain, or itching, or other metrics related to the alleviation of signs or symptoms of NASH, compared to an untreated subject. Methods for measuring the alleviation of signs and symptoms of NASH are known in the art.

[0214] [Table 8]

[0215] As will be readily apparent to those skilled in the art, various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention as described herein. The invention as described exemplary herein can be suitably implemented without any elements or limitations not specifically disclosed herein. The terms and expressions used herein are for illustrative purposes only, not limitations, and in the use of such terms and expressions there is no intention to exclude any equivalents of the illustrated and described features or parts thereof, but it should be recognized that various modifications are possible within the scope of the invention. Accordingly, although the invention is shown by specific embodiments and optional features, it should be understood that those skilled in the art can rely on modifications and / or changes to the concepts disclosed herein, and such modifications and changes will be considered within the scope of the invention.

[0216] This specification may refer to several patent and non-patent references. Any cited references are incorporated herein by reference in their entirety. If there is any inconsistency between the definition of a term in this specification and the definition in the cited references, that term should be interpreted based on the definition herein.

Claims

1. A modified Christensenella minuta bile salt hydrolase (BSH) enzyme that is at least 90% identical to SEQ ID NO: 1, and comprises 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 one or more of the 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 of the 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 one or more of the substitutions include I57V.

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

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

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

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

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

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

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

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

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

14. The modified enzyme according to claim 1, comprising one of sequence numbers 2 to 47.

15. The modified enzyme according to claim 1, comprising a sequence having at least 90% identity with SEQ ID NO: 5, having the substitution I57V in SEQ ID NO: 1, and optionally comprising or consisting of SEQ ID NO:

5.

16. The modified enzyme according to claim 1, comprising a sequence having at least 90% identity with sequence number 6, having the substitution Y64S in sequence number 1, and optionally comprising or consisting of sequence number 6.

17. The modified enzyme according to claim 1, comprising a sequence having at least 90% identity with sequence number 8, having the substitution S136A in sequence number 1, and optionally comprising or consisting of sequence number 8.

18. The modified enzyme according to claim 1, comprising a sequence having at least 90% identity with SEQ ID NO: 9, having the substitution K189P in SEQ ID NO: 1, and optionally comprising or consisting of SEQ ID NO:

9.

19. The modified enzyme according to claim 1, comprising a sequence having at least 90% identity with SEQ ID NO: 36, having the substitutions Y8V, P81A, S206G, and R207Q in SEQ ID NO: 1, and optionally comprising or consisting of SEQ ID NO:

36.

20. The modified enzyme according to claim 1, comprising a sequence having at least 90% identity with SEQ ID NO: 37, having the substitutions Y34K, I57V, L127I, and N292S in SEQ ID NO: 1, and optionally comprising or consisting of SEQ ID NO:

37.

21. The modified enzyme according to claim 1, comprising a sequence having at least 90% identity with SEQ ID NO: 41, having the substitutions Y56T, I57V, L127I, and N292S in SEQ ID NO: 1, and optionally comprising or consisting of SEQ ID NO:

41.

22. The modified enzyme according to claim 1, comprising a sequence having at least 90% identity with SEQ ID NO: 42, having the substitutions K90E, L127I, N292S, and R323E in SEQ ID NO: 1, and optionally comprising or consisting of SEQ ID NO:

42.

23. The modified enzyme according to claim 1, comprising a sequence having at least 90% identity with SEQ ID NO: 43, having the substitutions Y34N, H47N, Y64F, Y176K, A221P in SEQ ID NO: 1, and optionally comprising or consisting of SEQ ID NO:

43.

24. The modified enzyme according to claim 1, comprising a sequence having at least 90% identity with SEQ ID NO: 44, and having the substitutions Y34N, N46K, H47N, Y64F, Y176K, and A221L in SEQ ID NO: 1, and optionally comprising or consisting of SEQ ID NO:

44.

25. The modified enzyme according to claim 1, comprising a sequence having at least 90% identity with SEQ ID NO: 45, and having the substitutions Y8T, Y34N, Y56V, I57V, F99Y, L127I, S136A, K189N, N292S, and R323L in SEQ ID NO: 1, and optionally comprising or consisting of SEQ ID NO:

45.

26. In sequence number 1, positions 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, N7 9S, D82G, N83I, D85S, K87G, V89P, K90T, E91P, Y93K, D94E, I96V, F99Y, F101L, W104Y, Q108R, A110G, S113D, R116L, I117E, E120K, Q 121T, I122L, L124I, L127V, N128D, E131A, L133H, S136K, L138V, H139K, W140Y, Q145K, R146E, D147K, V151L, S153Q, F161Y, V165T, N 170G, 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, G252D, E255R, Q256N, Q257V, R258P, Modified bile salt hydrolase (BSH) enzymes, including amino acid substitutions of 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. In sequence number 1, positions 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, L63I, Y64F, Y65K, D66M, G77A, L78V, N79S, F80L, D82G, N83 I, D85S, K87G, V89K, K90T, Y93K, D94E, F99Y, F101L, 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, L200I, E201K, Q204R, Y205I, R207A, M209D, A211N, L218K, S220D, A221L, R223K, V225I, K226R , A228T, T230V, K231R, M232L, S234A, S239T, S241D, S243Q, I244L, S245M, G252D, E255R, Q256D, Q257V, R258P, C260T, H262W, E265G Modified bile salt hydrolase (BSH) enzymes, including amino acid substitutions of 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 sequence numbers 46 or 47.

29. The modified enzyme according to claim 1, which has increased activity at approximately pH 5 compared to wild-type C. minuta BSH.

30. The modified enzyme according to claim 1, which has increased activity at approximately pH 7 compared to wild-type C. minuta BSH.

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

32. The polynucleotide according to claim 31, comprising one of sequence numbers 48 to 79.

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

34. The polynucleotide according to claim 31, wherein one or more regulatory elements include a promoter and / or an enhancer.

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

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

37. The polynucleotide according to claim 33, wherein one or more regulatory elements are promoters, and the promoters are inductive promoters.

38. The polynucleotide according to claim 37, wherein the promoter is inducible by environmental conditions or responsive to hypoxic or anaerobic conditions.

39. The polynucleotide according to claim 37, comprising one of sequence numbers 104 to 109.

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

41. A pharmaceutical composition comprising the modified Christensenella minuta bile salt hydrolase (BSH) enzyme described in claim 1.

42. A manipulated bacterial cell comprising the polynucleotide described in claim 31.

43. The manipulated bacterial cell according to claim 42, wherein the expression of the polynucleotide is functionally bound to an exogenous promoter not found in the natural Christensenella minuta genome.

44. Asidaminococcus, Actinomyces, Ackermansia muciniphylla, Allobaculum, Anaerococcus, Anaerostipes, Bacteroides, Bacteroides and others, Bacteroides acidifaciens, Bacteroides coprofilus, Bacteroides fragilis, Bacteroides ovatus, Bacteroides uniformis, Barnesiellaceae, Bifidobacterium adolescentis, Bifidobacterium and others, Bifidobacterium, Bilophila, Brautia obeum, Brautia proda Kuta, Brautia and others, Brautia genus, Burraydia genus, Catenibacterium genus, Chrysenella genus, Citrobacter genus, Clostridiaceae genus, Clostridiales and others, Clostridiales genus, Clostridium perfringens, Clostridium genus, Clostridium and others, Chorinthera aerofasciens, Chorinthera genus, Chorinthera stercolis, Coprococcus catus, Coprococcus genus, Coriobacteriaceae genus, Desulfovibrio genus, Dialist genus, Dorea formisigenerans, Dorea genus, Dorea and others, Aegatacera lenta, Enterobacteria seaae and others, Enterobacteria seaae genus, Enterococcus genus, Erysipelotrichaceae genus, Eubacterium biforme, Eubacterium biforme, Eubacterium doricam, Eubacterium genus, Faecalibacterium prausnitzii, Fusobacterium genus, Gemeraceae genus, Haemophilus parainfluenzae, Haemophilus and others, Helicobacter genus, Helicobacter lachnospiraceae and others, Lachnospiraceae genus, Lactobacillus reuteri, Lactobacillus mucosae Lactobacillus zeae, Lactobacillus genus, Lactobacillus genus, Lactococcus genus, Leuconostocaceae genus, Megamonas genus, Megasphere genus, Metanobrevibacter genus, Mitsuokera marutafida, Mitsuokera genus, Musispirium sheldreli, Odolibacter genus, Osirospira genus, Parabacteroides distasonis, Parabacteroides genus, Paraprevotella genus, Paraprevotellaaceae genus, Parvimonas genus, Pediococcus genus, Pediococcus and others, Peptococcus genus, Peptoniphyllus genus, Peptostreptococcus anaerobius,Peptostreptococcus and others, Phascolarctobacterium, Prevotella copri, Prevotella, Prevotella stercorea, Prevotella family, Proteus, Lycenera family, Rosebria fesis, Rosebria, Ruminococcus family and others, Ruminococcus, Ruminococcus bromii, Ruminococcus gunavas, Ruminococcus, Ruminococcus and others, Ruminococcus turkes, Slachia, S24-7 spp., SMB53 spp. The manipulated bacterial cell according to claim 42, selected from the group consisting of Streptococcus anginosus, Streptococcus lutesiae, Streptococcus, other Streptococcus species, Stellera, Tulicibacter, UC Braidia, UC Enterobacteria seae, UC Faecalibacterium, UC Parabacteroides, UC Pediococcus, Baribaculum, Veillonella, Sattara, Tulicibacter, UC Clostridial, UC Erysipelotrichacea, UC Luminococcusea, Veillonella parbra, Veillonella, Veillonella dispa, and Weissella.

45. The bacterial cell according to claim 42, wherein the polynucleotide is incorporated into the genome of the bacterial cell.

46. A probiotic composition comprising the manipulated bacterial cells described in claim 42.

47. A pharmaceutical composition comprising the manipulated bacterial cells described in claim 42 or the modified BSH enzyme described in claim 1.

48. A method comprising administering the pharmaceutical composition described in claim 47 to a subject requiring it.

49. The method according to claim 48, wherein the subject requiring the method is suffering from one or more of the following: non-alcoholic fatty liver disease (NAFLD), diseases or disorders related to non-alcoholic fatty liver disease (NAFLD), metabolic syndrome, or cardiovascular disease.

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

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

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

53. A method for treating a cholestasis disorder in a subject requiring treatment for the cholestasis disorder, comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 47 to the subject to treat the cholestasis disorder in the subject.

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

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

56. A method for treating a disease or disorder related to metabolic syndrome in a subject requiring treatment for such a disease or disorder, comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 47 to the subject to treat the disease or disorder related to metabolic syndrome.

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

58. The method according to 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 to 58, wherein the required subject is an animal.

60. The method according to claim 59, wherein the required subject is a mammal.

61. The method according to claim 59, wherein the required subject is a dog, cat, cow, sheep, goat, chicken, turkey, pig, fish, crustacean, or mollusk.

62. The method according to claim 59, wherein the required subject is a human subject.

63. The method according to claim 59, wherein the subject requiring the method is a cat, and the cat is suffering from feline hepatic lipidosis.

64. A method comprising providing the probiotics described in claim 46 or the prebiotics described in claim 40 as a target.

65. A method for improving the yield of livestock animals, comprising providing an effective amount of the probiotics described in claim 46 to improve the yield of the livestock.

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

67. The method according to claim 66, wherein the livestock animal is a ruminant, a pig, a bird, a fish, a crustacean, or a mollusk.

68. The method according to claim 67, wherein the ruminant is selected from cattle, sheep, goats, deer, buffalo, or camels.

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

70. A medical food comprising the modified BSH enzyme described in claim 1, the prebiotic described in claim 40, or the bacterial cell described in claim 42.

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

72. The method according to claim 71, further comprising concentrating, purifying, or isolating the modified BSH enzyme.

73. A modified Christensenella minuta bile salt hydrolase (BSH) enzyme 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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