TOM ERIN (US)
HO CLEO (US)
SAVILE CHRISTOPHER (US)
KUMAR ABHINAV (US)
ESSER LAUREN (US)
CHAN ANDREA (US)
CLAY MICHAEL (US)
PIGULA ADRIANNA (US)
CHEN HSIANG-YUN (US)
WO2011147957A1 | 2011-12-01 |
US5166374A | 1992-11-24 | |||
US20120045807A1 | 2012-02-23 |
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CLAIMS 1. A genetically-modified cell capable of producing UDCA or a UDCA precursor comprising at least one heterologous polynucleotide encoding an enzyme involved in a metabolic pathway that converts sugar to UDCA or a UDCA precursor. 2. The cell of claim 1, comprising at least two heterologous polynucleotides, each encoding an enzyme involved in a metabolic pathway that converts sugar to UDCA or a UDCA precursor, wherein the encoded enzymes are operably connected along the metabolic pathway. 3. The cell of claim 1 or 2, wherein the UDCA precursor is desmosterol; cholesterol; 7- alpha-hydroxycholesterol; 7a-hydroxy-4-cholesten-3-one; 7a-hydroxy-5b-cholestan-3-one; 5b- cholestane-3a,7a-diol; (25R)-3a,7a-dihydroxy-5b-cholestanoic acid; (25R)-3a,7a-dihydroxy-5b- cholestanoyl-CoA; (25S)-3a,7a-dihydroxy-5b-cholestanoyl-CoA; (24E)-3a,7a-dihydroxy-5b- cholest-24-enoyl-CoA; 3a,7a-dihydroxy-24-oxo-5b-cholestanoyl-CoA; 3a,7a-dihydroxy-5b- cholan-24-oyl-CoA; 3a-hydroxy-7-oxo-5b-cholan-24-oyl-CoA; 3a,7b-dihydroxy-5b-cholan-24- oyl-CoA; 7a,12a-dihydroxy-4-cholesten-3-one; 7a,12a-dihydroxy-5b-cholestan-3-one; 5b- cholestane-3a,7a,12a-triol; (25R)-3a,7a,12a-trihydroxy-5b-cholestan-26-oic acid; (25R)- 3a,7a,12a-trihydroxy-5b-cholestanoyl-CoA; (25S)-3a,7a,12a-trihydroxy-5b-cholestanoyl-CoA; (24E)-3a,7a,12a-trihydroxy-5b-cholest-24-enoyl-CoA; 3a,7a,12a-trihydroxy-24-oxo-5b- cholestanoyl-CoA; 3a,7a,12a-trihydroxy-5b-cholan-24-oyl-CoA; or cholic acid. 4. The cell of any one of claims 1–3, wherein the encoded enzyme is DHCR7, DHCR24, CYP7A1, HSD3B7, CYP8B1, AKR1D1, AKR1C9, AKR1C4, CYP27A1, SLC27A5, FAT1, AMACR, ACOX2, POX1, HSD17B4, FOX2, SCP2, POT1, ERG10, 7a-HSD, 7b-HSD, or choloyl-CoA hydrolase. 5. The cell of any one of claims 1–4, wherein the encoded enzyme is involved in the metabolic pathway that converts sugar to cholesterol. 6. The cell of any one of claims 1–4, wherein the encoded enzyme is involved in the metabolic pathway that converts cholesterol to CDC-CoA. 7. The cell of any one of claims 1–4, wherein the encoded enzyme is involved in the metabolic pathway that converts cholesterol to cholic acid. 8. The cell of any one of claims 1–4, wherein the encoded enzyme is involved in the metabolic pathway that converts CDC-CoA to UDCA. 9. The cell of any one of claims 1–5, wherein the encoded enzyme is: DHCR7 and is encoded by a polynucleotide comprising a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 2, 4, 6, 8, 10, or 12; or DHCR24 and is encoded by a polynucleotide comprising a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 14, 15, 16, 18, 19, 20, 22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, 36, 38, 39, 40, 42, 44, 46, or 48. 10. The cell of any one of claims 1–4 or 6–7, wherein the encoded enzyme is: CYP7A1 and is encoded by a polynucleotide comprising a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, or 80; HSD3B7 and is encoded by a polynucleotide comprising a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 82, 84, 86, or 88; CYP8B1 and is encoded by a polynucleotide comprising a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 266, 268, 270, 272, 274, 276, or 278; AKR1D1 and is encoded by a polynucleotide comprising a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 90, 92, 94, or 96; AKR1C9 and is encoded by a polynucleotide comprising a nucleic acid sequence that is substantially similar to SEQ ID NO: 98; AKR1C4 and is encoded by a polynucleotide comprising a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, or 122; CYP27A1 and is encoded by a polynucleotide comprising a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 124, 126, 128, 130, 132, 134, 136, or 138; SLC27A5 and is encoded by a polynucleotide comprising a nucleic acid sequence that is substantially identical to SEQ ID NOs: 140 or 142; FAT1 and is encoded by a polynucleotide comprising a nucleic acid sequence that is substantially identical to SEQ ID NO: 144; AMACR and is encoded by a polynucleotide comprising a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 146, 148, 150, 152, 154, 156, or 158; ACOX2 and is encoded by a polynucleotide comprising a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 160, 162, 164, 166, 168, 170, 172, or 174; POX1 and is encoded by a polynucleotide comprising a nucleic acid sequence that is substantially identical to SEQ ID NO: 176; HSD17B4 and is encoded by a polynucleotide comprising a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 178, 180, 182, 184, 186, 188, 190, or 192; FOX2 and is encoded by a polynucleotide comprising a nucleic acid sequence that is substantially identical to SEQ ID NO: 194; SCP2 and is encoded by a polynucleotide comprising a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 196, 198, 200, or 202; POT1 and is encoded by a polynucleotide comprising a nucleic acid sequence that is substantially identical to SEQ ID NO: 204; or ERG10 and is encoded by a polynucleotide comprising a nucleic acid sequence that is substantially identical to SEQ ID NO: 206. 11. The cell of claim 8, wherein the encoded enzyme is: 7a-HSD and is encoded by a polynucleotide comprising a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 208, 210, 212, or 214; 7b-HSD is encoded by a polynucleotide comprising a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 216, 218, 220, or 222; and choloyl-CoA hydrolase is encoded by a polynucleotide comprising a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 224, 226, 228, or 230. 12. The cell of any one of claims 1–11, further comprising a heterologous polynucleotide encoding ADR, ADX, and/or a truncated HMG 13. The cell of any one of claims 1–12, wherein the cell is a microorganism or part of a microorganism. 14. The cell of any one of claims 1–13, wherein the cell is bacterium or a yeast. 15. The cell of any one of claims 1–14, wherein the cell is Saccharomyces cerevisiae. 16. A method of making UDCA or a UDCA precursor, the method comprising: (a) contacting a substrate with the genetically-modified cell of any one of claims 1– 15; and (b) growing the cell to make UDCA or UDCA precursor. 17. The method of claim 16, further comprising isolating the UDCA or UDCA precursor from the cell. 18. The use of UDCA or UDCA precursor made using the method of claim 16 or 17 for the manufacture of a medicament for the treatment of a disease or a symptom of a disease. 19. The use of claim 19, wherein the disease or symptom of a disease is gallstones, primary biliary cirrhosis, cystic fibrosis, impaired bile flow, intrahepatic cholestasis of pregnancy, and/or cholelithiasis. 20. A medicament comprising UDCA or UDCA precursor made using the method of claim 16 or 17. 21. A method of treating a disease or symptom of a disease comprising administering UDCA or a UDCA precursor made using the method of claim 15 or 16 to a subject in need thereof. 22. The method of claim 21 wherein the disease or symptom of a disease is gallstones, primary biliary cirrhosis, cystic fibrosis, impaired bile flow, intrahepatic cholestasis of pregnancy, and/or cholelithiasis. 23. An isolated polynucleotide encoding at least one enzyme involved in a metabolic pathway that converts sugar to UDCA or a UDCA precursor. 24. The polynucleotide of claim 23, wherein the encoded enzyme is DHCR7, DHCR24, CYP7A1, HSD3B7, CYP8B1, AKR1D1, AKR1C9, AKR1C4, CYP27A1, SLC27A5, FAT1, AMACR, ACOX2, POX1, HSD17B4, FOX2, SCP2, POT1, ERG10, 7a-HSD, 7b-HSD, or choloyl-CoA hydrolase. 25. The polynucleotide of claim 23 or 24, wherein the encoded enzyme is involved in the metabolic pathway that converts sugar to cholesterol. 26. The polynucleotide of claim 23 or 24, wherein the encoded enzyme is involved in the metabolic pathway that converts cholesterol to CDC-CoA. 27. The polynucleotide of claim 23 or 24, wherein the encoded enzyme is involved in the metabolic pathway that converts cholesterol to cholic acid. 28. The polynucleotide of claim 23 or 24, wherein the encoded enzyme is involved in the metabolic pathway that converts CDC-CoA to UDCA. 29. The polynucleotide of any one of claims 23–25, wherein the encoded enzyme is: DHCR7 and the polynucleotide comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 2, 4, 6, 8, 10, or 12; or DHCR24 and the polynucleotide comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 14, 15, 16, 18, 19, 20, 22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, 36, 38, 39, 40, 42, 44, 46, or 48. 30. The polynucleotide of any one of claims 23–24 and 26–27, wherein the encoded enzyme is: CYP7A1 and the polynucleotide comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, or 80; HSD3B7 and the polynucleotide comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 82, 84, 86, or 88; CYP8B1 and the polynucleotide comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 266, 268, 270, 272, 274, 276, or 278; AKR1D1 and the polynucleotide comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 90, 92, 94, or 96; AKR1C9 and the polynucleotide comprises a nucleic acid sequence that is substantially similar to SEQ ID NO: 98; AKR1C4 and the polynucleotide comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, or 122; CYP27A1 and the polynucleotide comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 124, 126, 128, 130, 132, 134, 136, or 138; SLC27A5 and the polynucleotide comprises a nucleic acid sequence that is substantially identical to SEQ ID NOs: 140 or 142; FAT1 and the polynucleotide comprises a nucleic acid sequence that is substantially identical to SEQ ID NO: 144; AMACR and the polynucleotide comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 146, 148, 150, 152, 154, 156, or 158; ACOX2 and the polynucleotide comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 160, 162, 164, 166, 168, 170, 172, or 174; POX1 and the polynucleotide comprises a nucleic acid sequence that is substantially identical to SEQ ID NO: 176; HSD17B4 and the polynucleotide comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 178, 180, 182, 184, 186, 188, 190, or 192; FOX2 and the polynucleotide comprises a nucleic acid sequence that is substantially identical to SEQ ID NO: 194; SCP2 and the polynucleotide comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 196, 198, 200, or 202; POT1 and the polynucleotide comprises a nucleic acid sequence that is substantially identical to SEQ ID NO: 204; or ERG10 and the polynucleotide comprises a nucleic acid sequence that is substantially identical to SEQ ID NO: 206. 31. The polynucleotide of any one of claims 23–24 and 28, wherein the encoded enzyme is: 7a-HSD and the polynucleotide comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 208, 210, 212, or 214; 7b-HSD and the polynucleotide comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 216, 218, 220, or 222; and choloyl-CoA hydrolase and the polynucleotide comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 224, 226, 228, or 230. 32. A vector comprising a nucleic acid encoding at least one enzyme involved in a metabolic pathway that converts sugar to UDCA or a UDCA precursor. 33. The vector of claim 32, wherein the encoded enzyme is DHCR7, DHCR24, CYP7A1, HSD3B7, CYP8B1, AKR1D1, AKR1C9, AKR1C4, CYP27A1, SLC27A5, FAT1, AMACR, ACOX2, POX1, HSD17B4, FOX2, SCP2, POT1, ERG10, 7a-HSD, 7b-HSD, or choloyl-CoA hydrolase. 34. The vector of claim 32 or 33, wherein the encoded enzyme is involved in the metabolic pathway that converts sugar to cholesterol. 35. The vector of claim 32 or 33, wherein the encoded enzyme is involved in the metabolic pathway that converts cholesterol to CDC-CoA. 36. The vector of claim 32 or 33, wherein the encoded enzyme is involved in the metabolic pathway that converts cholesterol to cholic acid. 37. The vector of claim 32 or 33, wherein the encoded enzyme is involved in the metabolic pathway that converts CDC-CoA to UDCA. 38. The vector of any one of claims 32–34, wherein the encoded enzyme is: DHCR7 and the vector comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 2, 4, 6, 8, 10, or 12; or DHCR24 and the vector comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 14, 15, 16, 18, 19, 20, 22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, 36, 38, 39, 40, 42, 44, 46, or 48. 39. The vector of any one of claims 32–33 and 35–36, wherein the encoded enzyme is: CYP7A1 and the vector comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, or 80; HSD3B7 and the vector comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 82, 84, 86, or 88; CYP8B1 and the vector comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 266, 268, 270, 272, 274, 276, or 278; AKR1D1 and the vector comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 90, 92, 94, or 96; AKR1C9 and the vector comprises a nucleic acid sequence that is substantially identical to SEQ ID NO: 98; AKR1C4 and the vector comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, or 122; CYP27A1 and the vector comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 124, 126, 128, 130, 132, 134, 136, or 138; SLC27A5 and the vector comprises a nucleic acid sequence that is substantially identical to SEQ ID NOs: 140 or 142; FAT1 and the vector comprises a nucleic acid sequence that is substantially identical to SEQ ID NO: 144; AMACR and the vector comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 146, 148, 150, 152, 154, 156, or 158; ACOX2 and the vector comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 160, 162, 164, 166, 168, 170, 172, or 174; POX1 and the vector comprises a nucleic acid sequence that is substantially identical to SEQ ID NO: 176; HSD17B4 and the vector comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 178, 180, 182, 184, 186, 188, 190, or 192; FOX2 and the vector comprises a nucleic acid sequence that is substantially identical to SEQ ID NO: 194; SCP2 and the vector comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 196, 198, 200, or 202; POT1 and the vector comprises a nucleic acid sequence that is substantially identical to SEQ ID NO: 204; or ERG10 and the vector comprises a nucleic acid sequence that s substantially identical to SEQ ID NO: 206. 40. The vector of any one of claims 32—33 and 37, wherein the encoded enzyme is: 7a-HSD and the vector comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 208, 210, 212, or 214; 7 -HSD and the vector comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 216, 218, 220, or 222; and choloyl-CoA hydrolase and the vector comprises a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 224, 226, 228, or 230. 41. A method of making a genetically-modified cell capable of synthesizing UDCA or a UDCA precursor, the method comprising: (a) contacting a cell with at least one heterologous polynucleotide encoding an enzyme involved in a metabolic pathway that converts sugar to UDCA or a UDCA precursor; and (b) growing the cell so that said polynucleotide is inserted into said microorganism. 42. The method of claim 41, wherein said cell is a bacterium or a yeast cell. 43. The method of claim 41 or 42, wherein the cell is a S accharomyces cereusiae cell. 44. A composition comprising UDCA or a UDCA precursor, a free acid or CoA thereof, or a pharmaceutically-acceptable derivative or prodrug thereof, the UDCA, UDCA precursor, free acid or CoA thereof, or pharmaceutically-acceptable derivative or prodrug thereof produced by a method of claim 16 or 17. |
Standard transfection techniques can be used to insert genes into a microorganism. As used herein, the term“transfection” or“transformation” can refer to the insertion of an exogenous nucleic acid or polynucleotide into a host cell. The exogenous nucleic acid or polynucleotide can be maintained as a non-integrated vector, for example, a plasmid or episomal vector, or alternatively, can be integrated into the host cell genome. The term transfecting or transfection is intended to encompass all conventional techniques for introducing nucleic acid or polynucleotide into cells/microorganisms. Examples of transfection techniques include, but are not limited to, lithium acetate mediated transformation, calcium phosphate precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection, rubidium chloride or polycation mediated transfection, protoplast fusion, and sonication. The transfection method that provides optimal transfection frequency and expression of the construct in the particular host cell line and type is favored. For stable transfectants, the constructs are integrated so as to be stably maintained within the host chromosome. In some cases, the preferred transfection is a stable transfection. In some cases, the integration of the gene occurs at a specific locus within the genome of the microorganism. Expression vectors or other nucleic acids can be introduced to selected cells/microorganisms by any of a number of suitable methods. For example, vector constructs can be introduced to appropriate cells by any of a number of transformation methods. Standard calcium chloride- mediated bacterial transformation is still commonly used to introduce naked DNA to bacteria (see, e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), but electroporation and conjugation can also be used (see, e.g., Ausubel et al., 1988, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y.). For the introduction of vector constructs to yeast or other fungal cells, chemical transformation and electroporation methods can be used (e.g., Rose et al., 1990, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Transformed cells can be isolated on selective media appropriate to the selectable marker used. Alternatively, or in addition, plates or filters lifted from plates can be scanned for GFP fluorescence to identify transformed clones. For the introduction of vectors comprising differentially expressed sequences to certain types of cells, the method used can depend on the form of the vector. Plasmid vectors can be introduced by any of a number of transfection methods, including, for example, lipid-mediated transfection (“lipofection”), DEAE-dextran-mediated transfection, electroporation or calcium phosphate precipitation (see, e.g., Ausubel et al., 1988, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y.). Lipofection reagents and methods suitable for transient transfection of a wide variety of transformed and non-transformed or primary cells are widely available, making lipofection an attractive method of introducing constructs to eukaryotic, and particularly mammalian cells in culture. Many companies offer kits and ways for this type of transfection. The host cell can be capable of expressing the construct encoding the desired protein, processing the protein and transporting a secreted protein to the cell surface for secretion. Processing includes co- and post-translational modification such as leader peptide cleavage, GPI attachment, glycosylation, ubiquitination, and disulfide bond formation. Cells/microorganisms can be transformed or transfected with the above-described expression vectors or polynucleotides coding for one or more enzymes as disclosed herein and cultured in nutrient media modified as appropriate for the specific cell/microorganism, inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. In some cases, electroporation methods can be used to deliver an expression vector. Expression of a vector (and the gene contained in the vector) can be verified by an expression assay, for example, qPCR, colony PCR, sequencing of a locus or whole genome sequencing, or by measuring levels of RNA. Expression level can be indicative also of copy number. For example, if expression levels are extremely high, this can indicate that more than one copy of a gene was integrated in a genome. Alternatively, high expression can indicate that a gene was integrated in a highly transcribed area, for example, near a highly expressed promoter. Expression can also be verified by measuring protein levels, such as through Western blotting. CRISPR/Cas system
The methods disclosed throughout can involve pinpoint insertion of genes or the deletion of genes (or parts of genes). Methods described herein can use a CRISPR/Cas system. For example, double-strand breaks (DSBs) can be generated using a CRISPR/Cas system, e.g., a type II CRISPR/Cas system. A Cas enzyme used in the methods disclosed herein can be Cas9, which catalyzes DNA cleavage. Enzymatic action by Cas9 from Streptococcus pyogenes or any closely related Cas9 can generate double stranded breaks at target site sequences which hybridize to 20 nucleotides of a guide sequence and have a protospacer-adjacent motif (PAM) following the 20 nucleotides of the target sequence. A vector can be operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein and Mad7. Cas proteins that can be used include class 1 and class 2. Non-limiting examples of Cas proteins include Cas1, Cas1B Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1 , Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, C2c1, C2c2, C2c3, Cpf1, CARF, DinG, homologues thereof, or modified versions thereof. An unmodified CRISPR enzyme can have DNA cleavage activity, such as Cas9. A CRISPR enzyme can direct cleavage of one or both strands at a target sequence, such as within a target sequence and/or within a complement of a target sequence. For example, a CRISPR enzyme can direct cleavage of one or both strands within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 300, 400, 500, or more base pairs from the first or last nucleotide of a target sequence. A vector that encodes a CRISPR enzyme that is mutated to with respect, to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence can be used. A vector that encodes a CRISPR enzyme comprising one or more nuclear localization sequences (NLSs) can be used. For example, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs used. A CRISPR enzyme can comprise the NLSs at or near the ammo-terminus (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs), or at or near the carboxy-terminus (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs), or any combination of these (e.g., one or more NLS at the ammo-terminus and one or more NLS at the carboxy terminus). When more than one NLS is present, each can be selected independently of others, such that a single NLS can be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies. CRISPR enzymes used in the methods can comprise at most 6 NLSs. An NLS is considered near the N- or C-terminus when the nearest amino acid to the NLS is within 50 amino acids along a polypeptide chain from the N- or C-terminus, e.g., within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, or 50 amino acids. Guide RNA
As used herein, the term“guide RNA” and its grammatical equivalents refers to an RNA that can specifically target a DNA sequence and form a complex with Cas protein. An RNA/Cas complex can assist in“guiding” Cas protein to a target DNA. A method disclosed herein also can comprise introducing into a cell or embryo at least one guide RNA or nucleic acid, e.g., DNA encoding at least one guide RNA. A guide RNA can interact with a RNA-guided endonuclease to direct the endonuclease to a specific target site, at which site the 5’ end of the guide RNA base pairs with a specific protospacer sequence in a chromosomal sequence. A guide RNA can comprise two RNAs, e.g., CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA). A guide RNA can sometimes comprise a single-chain RNA, or single guide RNA (sgRNA) formed by fusion of a portion (e.g., a functional portion) of crRNA and tracrRNA. A guide RNA can also be a dualRNA comprising a crRNA and a tracrRNA. Furthermore, a crRNA can hybridize with a target DNA. As discussed above, a guide RNA can be an expression product. For example, a DNA that encodes a guide RNA can be a vector comprising a sequence coding for the guide RNA. A guide RNA can be transferred into a cell or microorganism by transfecting the cell or microorganism with an isolated guide RNA or plasmid DNA comprising a sequence coding for the guide RNA and a promoter. A guide RNA can also be transferred into a cell or microorganism in other ways, such as using virus-mediated gene delivery. A guide RNA can be isolated. For example, a guide RNA can be transfected in the form of an isolated RNA into a cell or microorganism. A guide RNA can be prepared by in vitro transcription using any in vitro transcription system. A guide RNA can be transferred to a cell in the form of isolated RNA rather than in the form of plasmid comprising encoding sequence for a guide RNA. A guide RNA can comprise three regions: a first region at the 5’ end that can be complementary to a target site in a chromosomal sequence, a second internal region that can form a stem loop structure, and a third 3’ region that can be single-stranded. A first region of each guide RNA can also be different such that each guide RNA guides a fusion protein to a specific target site. Further, second and third regions of each guide RNA can be identical in all guide RNAs. A first region of a guide RNA can be complementary to sequence at a target site in a chromosomal sequence such that the first region of the guide RNA can base pair with the target site. In some cases, a first region of a guide RNA can comprise from 10 nucleotides to 25 nucleotides (i.e., from 10 nucleotides to 25 nucleotides; or 10 nucleotides to 25 nucleotides; or from 10 nucleotides to 25 nucleotides; or from 10 nucleotides to 25 nucleotides or more. For example, a region of base pairing between a first region of a guide RNA and a target site in a chromosomal sequence can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more nucleotides in length. Sometimes, a first region of a guide RNA can be 19, 20, or 21 nucleotides in length. A guide RNA can also comprise a second region that forms a secondary structure. For example, a secondary structure formed by a guide RNA can comprise a stem (or hairpin) and a loop. A length of a loop and a stem can vary. For example, a loop can range from 3 to 10 nucleotides in length, and a stem can range from 6 to 20 base pairs in length. A stem can comprise one or more bulges of 1 to 10 nucleotides. The overall length of a second region can range from 16 to 60 nucleotides in length. For example, a loop can be 4 nucleotides in length and a stem can be 12 base pairs. A guide RNA can also comprise a third region at the 3’ end that can be essentially single-stranded. For example, a third region is sometimes not complementary to any chromosomal sequence in a cell of interest and is sometimes not complementary to the rest of a guide RNA. Further, the length of a third region can vary. A third region can be more than 4 nucleotides in length. For example, the length of a third region can range from 5 to 60 nucleotides in length. A guide RNA can be introduced into a cell or embryo as an RNA molecule. For example, a RNA molecule can be transcribed in vitro and/or can be chemically synthesized. An RNA can be transcribed from a synthetic DNA molecule, e.g., a gBlocks ® gene fragment. A guide RNA can then be introduced into a cell or embryo as an RNA molecule. A guide RNA can also be introduced into a cell or embryo in the form of a non-RNA nucleic acid molecule, e.g., DNA molecule. For example, a DNA encoding a guide RNA can be operably linked to promoter control sequence for expression of the guide RNA in a cell or embryo of interest. A RNA coding sequence can be operably linked to a promoter sequence that is recognized by RNA polymerase III (Pol III). Plasmid vectors that can be used to express guide RNA include, but are not limited to, px330 vectors and px333 vectors. In some cases, a plasmid vector (e.g., px333 vector) can comprise two guide RNA-encoding DNA sequences. A DNA sequence encoding a guide RNA can also be part of a vector. Further, a vector can comprise additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcriptional termination sequences, etc.), selectable marker sequences (e.g., antibiotic resistance genes), origins of replication, and the like. A DNA molecule encoding a guide RNA can also be linear. A DNA molecule encoding a guide RNA can also be circular. When DNA sequences encoding an RNA-guided endonuclease and a guide RNA are introduced into a cell, each DNA sequence can be part of a separate molecule (e.g., one vector containing an RNA-guided endonuclease coding sequence and a second vector containing a guide RNA coding sequence) or both can be part of a same molecule (e.g., one vector containing coding (and regulatory) sequence for both an RNA-guided endonuclease and a guide RNA). Site-specific insertion
Insertion of the genes can be site-specific. For example, one or more genes can be inserted adjacent to a promoter. Genes can also be inserted into a neutral location in a genome such as into a non-coding region or elsewhere such that wild-type gene function remains intact. Modification of a targeted locus of a cell/microorganism can be produced by introducing DNA into cell/microorganisms, where the DNA has homology to the target locus. DNA can include a marker gene, allowing for selection of cells comprising the integrated construct. Homologous DNA in a target vector can recombine with DNA at a target locus. A marker gene can be flanked on both sides by homologous DNA sequences, a 3’ recombination arm, and a 5’ recombination arm. A variety of enzymes can catalyze insertion of foreign DNA into a microorganism genome. For example, site-specific recombinases can be clustered into two protein families with distinct biochemical properties, namely tyrosine recombinases (in which DNA is covalently attached to a tyrosine residue) and serine recombinases (where covalent attachment occurs at a serine residue). In some cases, recombinases can comprise Cre, FC31 integrase (a serine recombinase derived from Streptomyces phage FC31), or bacteriophage derived site-specific recombinases (including Flp, lambda integrase, bacteriophage HK022 recombinase, bacteriophage R4 integrase and phage TP901-1 integrase). The CRISPR/Cas system can be used to perform site specific insertion. For example, a nick on an insertion site in the genome can be made by CRISPR/Cas to facilitate the insertion of a transgene at the insertion site. The methods described herein, can utilize techniques that can be used to allow a DNA or RNA construct entry into a host cell include, but are not limited to, calcium phosphate/DNA coprecipitation, microinjection of DNA into a nucleus, electroporation, bacterial protoplast fusion with intact cells, transfection, lipofection, infection, particle bombardment, sperm mediated gene transfer, or any other technique. Certain aspects disclosed herein can utilize vectors (including the ones described above). Any plasmids and vectors can be used as long as they are replicable and viable in a selected host microorganism. Vectors known in the art and those commercially available (and variants or derivatives thereof) can be engineered to include one or more recombination sites for use in the methods. Vectors that can be used include, but not limited to eukaryotic expression vectors such as pRS, pBluSkII, pET, pFastBac, pFastBacHT, pFastBacDUAL, pSFV, and pTet-Splice (Invitrogen), pEUK-C1, pPUR, pMAM, pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA, and pYACneo (Clontech), pSVK3, pSVL, pMSG, pCH110, and pKK232-8 (Pharmacia, Inc.), pXT1, pSG5, pPbac, pMbac, pMClneo, and pOG44 (Stratagene, Inc.), and pYES2, pAC360, pBlueBa- cHis A, B, and C, pVL1392, pBlueBac111, pCDM8, pcDNA1, pZeoSV, pcDNA3, pREP4, pCEP4, and pEBVHis (Invitrogen, Corp.), and variants or derivatives thereof. These vectors can be used to express a gene or portion of a gene of interest. A gene or portion of a gene can be inserted by using known methods, such as restriction enzyme or PCR-based techniques. Fermentation In some embodiments, the cells/microorganisms useful in the present invention should be cultured in fermentation conditions that are appropriate to convert a substrate to UDCA, cholic acid, and/or another UDCA precursor. Reaction conditions that should be considered include temperature, media flow rate, pH, media redox potential, agitation rate, inoculum level, maximum substrate concentrations, rates of introduction of the substrate to the bioreactor to ensure that substrate level does not become limiting, maximum product concentrations to avoid product inhibition, gas flow, gas composition, aeration rate, bio-reactor design, and media composition. The optimum reaction conditions will depend partly on the particular cell/microorganism used. However, in some cases, it is preferred that the fermentation be performed at a pressure higher than ambient pressure. The use of pressurized systems can greatly reduce the volume of the bioreactor required, and consequently the capital cost of the fermentation equipment. In some cases, reactor volume can be reduced in linear proportion to increases in reactor operating pressure, i.e. bioreactors operated at 10 atmospheres of pressure need only be one tenth the volume of those operated at 1 atmosphere of pressure. Fermentation Conditions
In those embodiments in which the cell/microorganism is cultured in fermentation conditions, the pH of the culture media may be optimized based on the cell/microorganism used. For example, the pH used can range from 4 to 10. In other instances, the pH can be from 5 to 9; 6 to 8; 6.1 to 7.9; 6.2 to 7.8; 6.3 to 7.7; 6.4 to 7.6; 6.5 to 7.5; 6.6 to 7.4; or 5.5 to 7.5. For example, the pH can be from 6.6 to 7.4. In some cases, the pH can be from 5 to 9. In some cases, the pH can be from 6 to 8. In some cases, the pH can be from 6.1 to 7.9. In some cases, the pH can be from 6.2 to 7.8. In some cases, the pH can be from 6.3 to 7.7. In some cases, the pH can be from 6.4 to 7.6. In some cases, the pH can be from 6.5 to 7.5. In some instances the pH used for the fermentation can be greater than about 6. In some instances the pH used for the fermentation can be lower than about 10. Temperature can also be adjusted based on the cell/microorganism used. For example, the temperature can range from 27°C to 45°C; 28°C to 44°C; 29°C to 43°C; 30°C to 42°C; 31°C to 41°C; 32°C to 40°C; or 36°C to 39°C. Availability of oxygen and other gases may affect yield and fermentation rate. For example, when considering oxygen availability, the percent of dissolved oxygen (DO) within the fermentation media can be from 1% to 40%. In certain instances, the DO concentration can be from 1.5% to 35%; 2% to 30%; 2.5% to 25%; 3% to 20%; 4% to 19%; 5% to 18%; 6% to 17%; 7% to 16%; 8% to 15%; 9% to 14%; 10% to 13%; or 11% to 12%. For example, in some cases the DO concentration can be from 2% to 30%. In other cases, the DO can be from 3% to 20%. In some cases, the DO can be from 4% to 10%. In some cases, the DO can be from 1.5% to 35%. In some cases, the DO can be from 2.5% to 25%. In some cases, the DO can be from 4% to 19%. In some cases, the DO can be from 5% to 18%. In some cases, the DO can be from 6% to 17%. In some cases, the DO can be from 7% to 16%. In some cases, the DO can be from 8% to 15%. In some cases, the DO can be from 9% to 14%. In some cases, the DO can be from 10% to 13%. In some cases, the DO can be from 11% to 12%. In some cases, atmospheric CO 2 can help to control the pH within cell culture medium. pH contained within cell culture media is dependent on a balance of dissolved CO 2 and bicarbonate (HCO3). Changes in atmospheric CO 2 can alter the pH of the medium. In certain instances, the atmospheric CO 2 can be from 0% to 10%; 0.01% to 9%; 0.05% to 8%; 0.1% to 7%; 0.5% to 6%; 1% to 5%; 2% to 4%; 3% to 6%; 4% to 7%; 2% to 6%; or 5% to 10%. In cases where a switch is used, the media can comprise the molecule that induces or represses the switch. When a lanthanum switch is used to repress the expression of one or more of the genes described herein, the media can comprise lanthanum, which will repress expression of the one or more genes under the control of the switch. In the case of lanthanum any one of the following concentrations can effectively repress expression of the one or more genes: 0.1 µM; 0.5 µM; 1 µM; 2 µM; 3 µM; 4 µM; 5 µM; 6 µM; 7 µM; 8 µM; 9 µM; 10 µM; 12.5 µM; 15 µM; 17.5 µM; 20 µM; 25 µM; 50 µM; 100 µM or more. In one case, 0.1 µM lanthanum can be used to repression expression of the one or more genes under the control of a lanthanum switch. In other cases, at least 0.5 µM lanthanum can be used. In other cases, at least 1 µM lanthanum can be used. In other cases, at least 2 µM lanthanum can be used. In other cases, at least 3 µM lanthanum can be used. In other cases, at least 4 µM lanthanum can be used. In other cases, at least 5 µM lanthanum can be used. In other cases, at least 6 µM lanthanum can be used. In other cases, at least 7 µM lanthanum can be used. In other cases, at least 8 µM lanthanum can be used. In other cases, at least 9 µM lanthanum can be used. In other cases, at least 10 µM lanthanum can be used. In other cases, at least 12.5 µM lanthanum can be used. In other cases, at least 15 µM lanthanum can be used. In other cases, at least 17.5 µM lanthanum can be used. In other cases, at least 20 µM lanthanum can be used. In other cases, at least 25 µM lanthanum can be used. In other cases, at least 50 µM lanthanum can be used. In other cases, at least 100 µM lanthanum can be used. In some cases, a range of 0.5 µM lanthanum to 100 µM lanthanum will effectively repress gene expression. In some cases, a range of 0.5 µM lanthanum to 50 µM lanthanum will repress gene expression. In other cases, a range of 1 µM lanthanum to 20 µM lanthanum will repress gene expression. In some cases, a range of 2 µM lanthanum to 15 µM lanthanum will repress gene expression. In some cases, a range of 3 µM lanthanum to 12.5 µM lanthanum will repress gene expression. In some cases, a range of 4 µM lanthanum to 12 µM lanthanum will repress gene expression. In some cases, a range of 5 µM lanthanum to 11.5 µM lanthanum will repress gene expression. In some cases, a range of 6 µM lanthanum to 11 µM lanthanum will repress gene expression. In some cases, a range of 7 µM lanthanum to 10.5 µM lanthanum will repress gene expression. In some cases, a range of 8 µM lanthanum to 10 µM lanthanum will repress gene expression. In some cases, the lanthanum in the media can be diluted to turn on expression of the one or more lanthanum repressed genes. For example, in some cases, the dilution of lanthanum containing media can be 1:1 (1 part lanthanum containing media to 1 part non-lanthanum containing media). In some cases, the dilution can be at least 1:2; 1:3; 1:4; 1:5; 1:7.5; 1:10; 1:15; 1:20; 1:25; 1:30; 1:35; 1:40; 1:45; 1:50; 1:75; 1:100; 1:200; 1:300; 1:400; 1:500; 1:1,000; or 1:10,000. For example, in some cases, a 1:2 dilution can be used. In some cases, at least a 1:3 dilution can be used. In some cases, at least a 1:4 dilution can be used. In some cases, at least a 1:5 dilution can be used. In some cases, at least a 1:7.5 dilution can be used. In some cases, at least a 1:10 dilution can be used. In some cases, at least a 1:15 dilution can be used. In some cases, at least a 1:20 dilution can be used. In some cases, at least a 1:25 dilution can be used. In some cases, at least a 1:30 dilution can be used. In some cases, at least a 1:35 dilution can be used. In some cases, at least a 1:40 dilution can be used. In some cases, at least a 1:45 dilution can be used. In some cases, at least a 1:50 dilution can be used. In some cases, at least a 1:75 dilution can be used. In some cases, at least a 1:100 dilution can be used. In some cases, at least a 1:200 dilution can be used. In some cases, at least a 1:300 dilution can be used. In some cases, at least a 1:400 dilution can be used. In some cases, at least a 1:500 dilution can be used. In some cases, at least a 1:1,000 dilution can be used. In some cases, at least a 1:10,000 dilution can be used. In some cases, the cell/microorganism may be grown in media comprising lanthanum. The media can then be diluted to effectively turn on the expression of the lanthanum repressed genes. The cell/microorganism can be then grown in conditions to promote the production of desired products, such as UDCA, cholic acid, and/or other UDCA precursors (as disclosed throughout). When a glucose to galactose switch is used to repress the expression of one or more of the genes described herein (e.g., when a GAL1 or GAL10 promoter is used), the media can comprise glucose, which will repress expression of the one or more genes under the control of the switch. In the case of glucose any one of the following concentrations can effectively repress expression of the one or more genes: 0.1 %; 0.5 %; 1 %; 2 %; 3 %; 4 %; 5 %; 6 %; 7 %; 8 %; 9 %; 10 %; 12.5 %; 15 %; 17.5 %; 20 %; 25 %; 50 %; 100 % or more. In one case, 0.1 % glucose can be used to repression expression of the one or more genes under the control of a glucose to galactose switch. In other cases, at least 0.5 % glucose can be used. In other cases, at least 1 % glucose can be used. In other cases, at least 2 % glucose can be used. In other cases, at least 3 % glucose can be used. In other cases, at least 4 % glucose can be used. In other cases, at least 5 % glucose can be used. In other cases, at least 6 % glucose can be used. In other cases, at least 7 % glucose can be used. In other cases, at least 8 % glucose can be used. In other cases, at least 9 % glucose can be used. In other cases, at least 10 % glucose can be used. In other cases, at least 12.5 % glucose can be used. In other cases, at least 15 % glucose can be used. In other cases, at least 17.5 % glucose can be used. In other cases, at least 20 % glucose can be used. In other cases, at least 25 % glucose can be used. In other cases, at least 50 % glucose can be used. In other cases, at least 100 % glucose can be used. In some cases, a range of 0.5 % glucose to 100 % glucose will effectively repress gene expression. In some cases, a range of 0.5 % glucose to 50 % glucose will repress gene expression. In other cases, a range of 1 % glucose to 20 % glucose will repress gene expression. In some cases, a range of 2 % glucose to 15 % glucose will repress gene expression. In some cases, a range of 3 % glucose to 12.5 % glucose will repress gene expression. In some cases, a range of 4 % glucose to 12 % glucose will repress gene expression. In some cases, a range of 5 % glucose to 11.5 % glucose will repress gene expression. In some cases, a range of 6 % glucose to 11 % glucose will repress gene expression. In some cases, a range of 7 % glucose to 10.5 % glucose will repress gene expression. In some cases, a range of 8 % glucose to 10 % glucose will repress gene expression. In some cases, the glucose in the media can be diluted to turn on expression of the one or more glucose repressed genes. For example, in some cases, the dilution of glucose containing media can be 1:1 (1 part glucose containing media to 1 part non-glucose containing media). In some cases, the dilution can be at least 1:2; 1:3; 1:4; 1:5; 1:7.5; 1:10; 1:15; 1:20; 1:25; 1:30; 1:35; 1:40; 1:45; 1:50; 1:75; 1:100; 1:200; 1:300; 1:400; 1:500; 1:1,000; or 1:10,000. For example, in some cases, a 1:2 dilution can be used. In some cases, at least a 1:3 dilution can be used. In some cases, at least a 1:4 dilution can be used. In some cases, at least a 1:5 dilution can be used. In some cases, at least a 1:7.5 dilution can be used. In some cases, at least a 1:10 dilution can be used. In some cases, at least a 1:15 dilution can be used. In some cases, at least a 1:20 dilution can be used. In some cases, at least a 1:25 dilution can be used. In some cases, at least a 1:30 dilution can be used. In some cases, at least a 1:35 dilution can be used. In some cases, at least a 1:40 dilution can be used. In some cases, at least a 1:45 dilution can be used. In some cases, at least a 1:50 dilution can be used. In some cases, at least a 1:75 dilution can be used. In some cases, at least a 1:100 dilution can be used. In some cases, at least a 1:200 dilution can be used. In some cases, at least a 1:300 dilution can be used. In some cases, at least a 1:400 dilution can be used. In some cases, at least a 1:500 dilution can be used. In some cases, at least a 1:1,000 dilution can be used. In some cases, at least a 1:10,000 dilution can be used. In cases where a switch is used, the media can comprise the molecule that de-represses the switch. For example, when a glucose to galactose switch is used to repress the expression of one or more of the genes described herein (e.g., when a GAL1 or GAL10 promoter is used), the media can comprise raffinose, which will de-repress expression of the one or more genes under the control of the switch. In the case of raffinose any one of the following concentrations can effectively repress expression of the one or more genes: 0.1 %; 0.5 %; 1 %; 2 %; 3 %; 4 %; 5 %; 6 %; 7 %; 8 %; 9 %; 10 %; 12.5 %; 15 %; 17.5 %; 20 %; 25 %; 50 %; 100 % or more. In one case, 0.1 % raffinose can be used to de-repress expression of the one or more genes under the control of a raffinose switch. In other cases, at least 0.5 % raffinose can be used. In other cases, at least 1 % raffinose can be used. In other cases, at least 2 % raffinose can be used. In other cases, at least 3 % raffinose can be used. In other cases, at least 4 % raffinose can be used. In other cases, at least 5 % raffinose can be used. In other cases, at least 6 % raffinose can be used. In other cases, at least 7 % raffinose can be used. In other cases, at least 8 % raffinose can be used. In other cases, at least 9 % raffinose can be used. In other cases, at least 10 % raffinose can be used. In other cases, at least 12.5 % raffinose can be used. In other cases, at least 15 % raffinose can be used. In other cases, at least 17.5 % raffinose can be used. In other cases, at least 20 % raffinose can be used. In other cases, at least 25 % raffinose can be used. In other cases, at least 50 % raffinose can be used. In other cases, at least 100 % raffinose can be used. In some cases, a range of 0.5 % raffinose to 100 % raffinose will effectively repress gene expression. In some cases, a range of 0.5 % raffinose to 50 % raffinose will de-repress gene expression. In other cases, a range of 1 % raffinose to 20 % raffinose will repress gene expression. In some cases, a range of 2 % raffinose to 15 % raffinose will repress gene expression. In some cases, a range of 3 % raffinose to 12.5 % raffinose will de-repress gene expression. In some cases, a range of 4 % raffinose to 12 % raffinose will de-repress gene expression. In some cases, a range of 5 % raffinose to 11.5 % raffinose will de-repress gene expression. In some cases, a range of 6 % raffinose to 11 % raffinose will de- repress gene expression. In some cases, a range of 7 % raffinose to 10.5 % raffinose will de-repress gene expression. In some cases, a range of 8 % raffinose to 10 % raffinose will de-repress gene expression. In cases where a switch is used, the media can comprise the molecule that induces the switch. For example, when a glucose to galactose switch is used to induce the expression of one or more of the genes (e.g., when a GAL1 or GAL10 promoter is used), the media can comprise galactose, which will induce expression of the one or more genes under the control of the switch. In the case of galactose any one of the following concentrations can effectively induce expression of the one or more genes: 0.1 %; 0.5 %; 1 %; 2 %; 3 %; 4 %; 5 %; 6 %; 7 %; 8 %; 9 %; 10 %; 12.5 %; 15 %; 17.5 %; 20 %; 25 %; 50 %; 100 % or more. In one case, 0.1 % galactose can be used to induce expression of the one or more genes under the control of a glucose to galactose switch. In other cases, at least 0.5 % galactose can be used. In other cases, at least 1 % galactose can be used. In other cases, at least 2 % galactose can be used. In other cases, at least 3 % galactose can be used. In other cases, at least 4 % galactose can be used. In other cases, at least 5 % galactose can be used. In other cases, at least 6 % galactose can be used. In other cases, at least 7 % galactose can be used. In other cases, at least 8 % galactose can be used. In other cases, at least 9 % galactose can be used. In other cases, at least 10 % galactose can be used. In other cases, at least 12.5 % galactose can be used. In other cases, at least 15 % galactose can be used. In other cases, at least 17.5 % galactose can be used. In other cases, at least 20 % galactose can be used. In other cases, at least 25 % galactose can be used. In other cases, at least 50 % galactose can be used. In other cases, at least 100 % galactose can be used. In some cases, a range of 0.5 % galactose to 100 % galactose will effectively induce gene expression. In some cases, a range of 0.5 % galactose to 50 % galactose will induce gene expression. In other cases, a range of 1 % galactose to 20 % galactose will induce gene expression. In some cases, a range of 2 % galactose to 15 % galactose will induce gene expression. In some cases, a range of 3 % galactose to 12.5 % galactose will induce gene expression. In some cases, a range of 4 % galactose to 12 % galactose will induce gene expression. In some cases, a range of 5 % galactose to 11.5 % galactose will induce gene expression. In some cases, a range of 6 % galactose to 11 % galactose will induce gene expression. In some cases, a range of 7 % galactose to 10.5 % galactose will induce gene expression. In some cases, a range of 8 % galactose to 10 % galactose will induce gene expression. When a copper switch is used to induce the expression of one or more of the genes described herein, the media can comprise copper, which will induce expression of the one or more genes under the control of the switch. In the case of copper any one of the following concentrations can effectively induce expression of the one or more genes: 1 µM; 2.5 µM; 5 µM; 10 µM; 25 µM; 50 µM; 75 µM; 100 µM; 150 µM; 200 µM; 300 µM; 400 µM; 500 µM; 600 µM; 700 µM; 800 µM; 900 µM; 1 mM; 10 mM or more. In one case, 1 µM copper can be used to induce expression of the one or more genes under the control of a copper promoter. In other cases, at least 5 µM copper can be used. In other cases, at least 10 µM copper can be used. In other cases, at least 25 µM copper can be used. In other cases, at least 50 µM copper can be used. In other cases, at least 100 µM copper can be used. In other cases, at least 200 µM copper can be used. In other cases, at least 300 µM copper can be used. In other cases, at least 400 µM copper can be used. In other cases, at least 500 µM copper can be used. In other cases, at least 600 µM copper can be used. In other cases, at least 700 µM copper can be used. In other cases, at least 800 µM copper can be used. In other cases, at least 900 µM copper can be used. In other cases, at least 1 mM copper can be used. In other cases, at least 2.5 mM copper can be used. In other cases, at least 5 mM copper can be used. In other cases, at least 7.5 mM copper can be used. In other cases, at least 10 mM copper can be used. In some cases, a range of 1 µM copper to 10 mM copper will effectively repress gene expression. In some cases, a range of 2.5 µM copper to 1 mM copper will repress gene expression. In other cases, a range of 5 µM copper to 800 µM copper will repress gene expression. In some cases, a range of 10 µM copper to 600 µM copper will repress gene expression. In some cases, a range of 25 µM copper to 500 µM copper will repress gene expression. In some cases, a range of 50 µM copper to 450 µM copper will repress gene expression. In some cases, a range of 75 µM copper to 400 µM copper will repress gene expression. In some cases, a range of 100 µM copper to 350 µM copper will repress gene expression. In some cases, a range of 150 µM copper to 300 µM copper will repress gene expression. In some cases, a range of 200 µM copper to 250 µM copper will repress gene expression. Bioreactor Fermentation reactions can be carried out in any suitable bioreactor. In some cases, the bioreactor can comprise a first, growth reactor in which the cells/microorganisms are cultured, and a second, fermentation reactor, to which broth from the growth reactor is fed and in which most of the fermentation product is produced. Product Recovery
The fermentation of the cells/microorganisms disclosed herein can produce a broth comprising a desired product (e.g., UDCA, cholic acid, and/or other UDCA precursor), one or more by- products, and/or the cell/microorganism itself. In certain methods of producing products, the concentration of products in the fermentation broth is at least 0.1 g/L. For example, the concentration of products produced in the fermentation broth can be from 0.1 g/L to 0.5 g/L, 0.5 g/L to 1 g/L, 1 g/L to 5 g/L, 2 g/L to 6 g/L, 3 g/L to 7 g/L, 4 g/L to 8 g/L, 5 g/L to 9 g/L, or 6 g/L to 10 g/L. In some cases, the concentration of products can be at least 9 g/L. In some cases, the concentration of products can be from 0.1 g/L to 10 g/L. In some cases, the concentration of products can be from 0.5 g/L to 3 g/L. In some cases, the concentration of products can be from 1 g/L to 5 g/L. In some cases, the concentration of products can be from 2 g/L to 6 g/L. In some cases, the concentration of products can be from 3 g/L to 7 g/L. In some cases, the concentration of products can be from 4 g/L to 8 g/L. In some cases, the concentration of products can be from 5 g/L to 9 g/L. In some cases, the concentration of products can be from 6 g/L to 10 g/L. In some cases, the concentration of products can be from 1 g/L to 3 g/L. In some cases, the concentration of products can be about 2 g/L. As discussed above, in certain cases the product produced in the fermentation reaction is converted to a different organic product. For example, the product produced may be a UDCA precursor that serves as a substrate for the further production of UDCA, cholic acid, or another UDCA precursor. In other cases, the product is first recovered from the fermentation broth before conversion to a different organic product. In some cases, the product can be continuously removed from a portion of broth and recovered as purified. In particular cases, the recovery of the product includes passing the removed portion of the broth containing the product through a separation unit to separate the cells/microorganisms from the broth, to produce a cell-free product permeate, and returning the microorganisms to the bioreactor. The cell-free product containing permeate can then can be stored or be used for subsequent conversion to a different desired product. The recovering of the desired product and/or one or more other products or by-products produced in the fermentation reaction can comprise continuously removing a portion of the broth and recovering separately the product and one or more other products from the removed portion of the broth. In some cases, the recovery of the product and/or one or more other products includes passing the removed portion of the broth containing the product and/or one or more other products through a separation unit to separate cells/microorganisms from the product and/or one or more other products, to produce a cell-free product and one or more other product-containing permeate, and returning the microorganisms to the bioreactor. In the above cases, the recovery of the product and one or more other products can include first removing the product from the cell-free permeate followed by removing the one or more other products from the cell-free permeate. The cell-free permeate can also then returned to the bioreactor. The product, or a mixed product stream containing the product, can be recovered from the fermentation broth. For example, methods that can be used can include but are not limited to, fractional distillation or evaporation, pervaporation, and extractive fermentation. Further examples include: recovery using steam from whole fermentation broths; reverse osmosis combined with distillation; liquid-liquid extraction techniques involving solvent extraction of the product; aqueous two-phase extraction of the product in PEG/dextran system; solvent extraction using alcohols or esters, e.g., ethyl acetate, tributylphosphate, diethyl ether, n-butanol, dodecanol, oleyl alcohol, and an ethanol/phosphate system; aqueous two-phase systems composed of hydrophilic solvents and inorganic salts. See generally, Voloch, M., et al., (1985) and U.S. Pat. Pub. Appl. No.2012/0045807. In some cases, the product and/or other by-products may be recovered from the fermentation broth by continuously removing a portion of the broth from the bioreactor, separating microbial cells from the broth (conveniently by filtration, for example), and recovering the product and others such as alcohols and acids from the broth. Alcohols can conveniently be recovered for example by distillation, and acids can be recovered for example by adsorption on activated charcoal. The separated microbial cells are returned to the fermentation bioreactor. The cell-free permeate remaining after the alcohol(s) and acid(s) have been removed is also preferably returned to the fermentation bioreactor. Additional nutrients can be added to the cell-free permeate to replenish the nutrient medium before it is returned to the bioreactor. Also, if the pH of the broth is adjusted during recovery of the product and/or by-products, the pH should be re-adjusted to a similar pH to that of the broth in the fermentation bioreactor, before being returned to the bioreactor. In Vitro Methods and Steps In some embodiments, the present invention relates in part to an in vitro method of making UDCA or UDCA precursor. In other words, in these embodiments, the method does not involve the use of a microorganism. For example, the substrate may be contacted with an enzyme or a fragment thereof, such as described previously, in a medium. In some embodiments, the method involves both in vivo and in vitro steps. For example, some reactions along the biosynthetic pathway can occur within a cell, whereas some of the reactions along the pathway occur outside of a cell. In certain such methods, a UDCA precursor may be secreted by a cell into media and then directly converted enzymatically or non-enzymatically (e.g., chemically) into a different product, such as UDCA or another DCA precursor. CoEnzyme A
The microorganism and methods described throughout can be used to produce a CoA-form of the products described throughout. In some cases, a CoA ligase can be used to produce a CoA form of any of the products described throughout. In some cases, SLC27A5 can produce a CoA product that is (25R)-3a,7a-dihydroxy-5b- cholestanoyl-CoA or (25R)-3a,7a,12a-trihydroxy-5b-cholestanoyl-CoA. In some cases, AMACR can produce a CoA product that is (25S)-3a,7a-dihydroxy-5b-cholestanoyl-CoA or (25S)- 3a,7a,12a-trihydroxy-5b-cholestanoyl-CoA. In some cases, ACOX2 can produce a CoA product that is (24E)-3a,7a-dihydroxy-5b-cholest-24-enoyl-CoA or (24E)-3a,7a,12a-trihydroxy-5b- cholest-24-enoyl-CoA. In some cases, HSD17B4 can produce a CoA product that is 3a,7a- dihydroxy-24-oxo-5b-cholestanoyl-CoA or 3a,7a,12a-trihydroxy-24-oxo-5b-cholestanoyl-CoA. In some cases, SCP2/Thiolase can produce a CoA product that is 3a,7a-dihydroxy-5b-cholan-24- oyl-CoA (CDC-CoA) or 3a,7a,12a-trihydroxy-5b-cholan-24-oyl-CoA. In some cases, 7a-HSD can produce a CoA product that is 3a-hydroxy-7-oxo-5b-cholan-24-oyl-CoA. In some cases, 7b- HSD can produce a CoA product that is 3a,7b-dihydroxy-5b-cholan-24-oyl-CoA (UDC-CoA). In some cases, the CoA form of one or more of the products can be (25R)-3a,7a-dihydroxy-5b- cholestanoyl-CoA; (25R)-3a,7a,12a-trihydroxy-5b-cholestanoyl-CoA; (25S)-3a,7a-dihydroxy-5b- cholestanoyl-CoA; (25S)-3a,7a,12a-trihydroxy-5b-cholestanoyl-CoA; (24E)-3a,7a-dihydroxy-5b- cholest-24-enoyl-CoA; (24E)-3a,7a,12a-trihydroxy-5b-cholest-24-enoyl-CoA; 3a,7a-dihydroxy- 24-oxo-5b-cholestanoyl-CoA; 3a,7a,12a-trihydroxy-24-oxo-5b-cholestanoyl-CoA; 3a,7a- dihydroxy-5b-cholan-24-oyl-CoA (CDC-CoA); 3a,7a,12a-trihydroxy-5b-cholan-24-oyl-CoA; 3a- hydroxy-7-oxo-5b-cholan-24-oyl-CoA; 3a,7b-dihydroxy-5b-cholan-24-oyl-CoA (UDC-CoA); or any combination thereof. The products as disclosed throughout can be isolated in their CoA form. Free Acids
The microorganism and methods described throughout can be used to produce a free acid-form of the products described throughout. In some cases, a hydrolase can be used to produce a free acid form of any of the products described throughout. In some cases, CYP27A1 can produce a free acid product that is (25R)-3a,7a-dihydroxy-5b- cholestanoic acid or (25R)-3a,7a,12a-trihydroxy-5b-cholestan-26-oic acid. In some cases, SLC27A5 can produce a free acid product that is (25R)-3a,7a-dihydroxy-5b-cholestanoic acid or (25R)-3a,7a,12a-trihydroxy-5b-cholestan-26-oic acid. In some cases, AMACR can produce a free acid product that is (25S)-3a,7a-dihydroxy-5b-cholestanoic acid or (25S)-3a,7a,12a-trihydroxy-5b- cholestanoic acid. In some cases, ACOX2 can produce a free acid product that is (24E)-3a,7a- dihydroxy-5b-cholest-24-enoic acid or (24E)-3a,7a,12a-trihydroxy-5b-cholest-24-enoic acid. In some cases, HSD17B4 can produce a free acid product that is 3a,7a-dihydroxy-24-oxo-5b- cholestanoic acid or 3a,7a,12a-trihydroxy-24-oxo-5b-cholestanoic acid. In some cases, SCP2/Thiolase can produce a free acid product that is 3a,7a-dihydroxy-5b-cholanoic acid (chenodeoxycholic acid; CDCA) or 3a,7a,12a-trihydroxy-5b-cholan-24-oic acid (cholic acid). In some cases, 7a-HSD can produce a free acid product that is 3a-hydroxy-7-oxo-5b-cholanoic acid (nutriacholic acid; NCA). In some cases, 7b-HSD can produce a free acid product that is 3a,7b- dihydroxy-5b-cholanoic acid (ursodeoxycholic acid; UDCA). In some cases, Choloyl-CoA hydrolase can produce a free acid product that is UDCA or 3a,7a,12a-trihydroxy-5b-cholan-24- oic acid (cholic acid). In some cases, the free acid form of one or more of the products can be (25R)-3a,7a-dihydroxy- 5b-cholestanoic acid; (25R)-3a,7a,12a-trihydroxy-5b-cholestan-26-oic acid; (25R)-3a,7a- dihydroxy-5b-cholestanoic acid; (25R)-3a,7a,12a-trihydroxy-5b-cholestan-26-oic acid; (25S)- 3a,7a-dihydroxy-5b-cholestanoic acid; (25S)-3a,7a,12a-trihydroxy-5b-cholestanoic acid; (24E)- 3a,7a-dihydroxy-5b-cholest-24-enoic acid; (24E)-3a,7a,12a-trihydroxy-5b-cholest-24-enoic acid; 3a,7a-dihydroxy-24-oxo-5b-cholestanoic acid; 3a,7a,12a-trihydroxy-24-oxo-5b-cholestanoic acid; 3a,7a-dihydroxy-5b-cholanoic acid (chenodeoxycholic acid; CDCA); 3a,7a,12a-trihydroxy-5b- cholan-24-oic acid (cholic acid); 3a-hydroxy-7-oxo-5b-cholanoic acid (nutriacholic acid; NCA); 3a,7b-dihydroxy-5b-cholanoic acid (ursodeoxycholic acid; UDCA); 3a,7a,12a-trihydroxy-5b- cholan-24-oic acid (cholic acid); or any combination thereof. The products as disclosed throughout can be isolated in their free acid form. Compositions The present invention also relates in part to a composition comprising UDCA or UDCA precursor, a free acid or CoA thereof, or a pharmaceutically-acceptable derivative or prodrug thereof. The composition may further comprise an excipient. The composition may be in the form of a medicament. A“pharmaceutically acceptable derivative” means any pharmaceutically acceptable salt, ester, salt of an ester, pro-drug or other derivative thereof. Pharmaceutically acceptable salts of the compounds of this invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate and undecanoate. Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl) +
4 salts. The present invention also relates in part to a method of formulating the UDCA or UDCA precursor into a pharmaceutical composition. For preparing pharmaceutical compositions from the compounds of the present invention, pharmaceutically-acceptable carriers include either solid or liquid carriers. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances, which also acts as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington’s Pharmaceutical Sciences, Maack Publishing Co, Easton PA. In powders, the carrier is a finely divided solid, which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired. Suitable solid excipients are carbohydrate or protein fillers include, but are not limited to sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; as well as proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents are added, such as the cross- linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution. The pharmaceutical preparation can be a unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. The present invention also relates to a method of making the pharmaceutical composition. In some cases, UDCA or a UDCA precursor is mixed with an excipient to produce a pharmaceutical composition. Treatment of Disease and Symptoms of Disease The UDCA or UDCA precursors (or other free acids or CoA products as disclosed throughout) can be used to treat disease. This includes treating one or more symptoms of the diseases. For example, the UDCA or a UDCA precursor (or other free acids or CoA products as disclosed throughout) can be used to treat one of more of the following diseases: gallstones (e.g., cholesterol gallstones), primary biliary cirrhosis, cystic fibrosis, impaired bile flow, intrahepatic cholestasis of pregnancy, and/or cholelithiasis. Some of the diseases or symptom of disease can be exclusive to humans, but other diseases or symptom of disease can be shared in more than one animal, such as in all mammals. The present invention relates in part to a method of treating a disease or symptom of a disease, the method comprising administering UDCA or UDCA precursor, a free acid or CoA thereof, or a pharmaceutically-acceptable derivative or prodrug thereof, to a subject in need of such treatment.
Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ophthalmic, pulmonary, transmucosal, transdermal, vaginal, otic, nasal, and topical administration. In addition, by way of example only, parenteral delivery includes intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intralymphatic, and intranasal injections.
Use of UDCA or UDCA precursor The present invention further relates in part to the use of the UDCA or UDCA precursor made using the aforementioned method in the manufacture of a medicament for the treatment or a disease or symptom of a disease. The disease or symptom of a disease may be any disease or symptom capable of being treated by UDCA or the UDCA precursor. Examples of such include gallstones, primary biliary cirrhosis, cystic fibrosis, impaired bile flow, intrahepatic cholestasis of pregnancy, and cholelithiasis.
UDCA can be used to treat gallstones and is a byproduct of intestinal bacteria.
The UDCA precursors may be used to make other products, such as other UDCA precursors or UDCA.
EXAMPLES
While some cases have been shown and described herein, such cases are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the cases of the invention described herein will be employed in practicing the invention. Example 1– Identification of enzymes that convert sugar to UDCA and generating strains that can make UDCA Thirteen heterologous enzymes (from the perspective of a Saccharomyces cerevisiae) were identified as possible enzymes that could be used to make UDCA from cholesterol. See e.g., FIG. 1. Two (2) additional enzymes were also identified as possible enzymes that could be used to convert sugar to cholesterol. See e.g., FIG.2. Genes encoding these enzymes were synthesized and then cloned into either yeast expression plasmids or into integration constructs. These plasmids or integrations constructs were subsequently transformed into Saccharomyces cerevisiae using standard yeast chemical transformation protocol, utilizing Lithium Acetate and PEG (3350). The transformed yeast were grown to mid log phase, then centrifuged at 4000 rpm with the supernatant removed. Pellets were washed with water and centrifuged again. The resulting pellet was resuspended in master mix containing 100 mM Lithium Acetate, 40% PEG (MW 3,350), 0.35 mg/ml carrier DNA (sheared salmon sperm DNA), and 50 to 500 ng of DNA to be transformed. The cell suspension was then incubated at 30˚C for 30 minutes, followed by at 45 minute heat shock at 42˚C. At this point, nutritional selection was plated, while antifungal selection underwent a 4 hr to overnight recovery in rich yeast media before plating on agar containing the antifungal drug. Plates were then incubated at 30˚C for 2 to 3 days. After colonies were formed, proper integrations were verified by colony PCR before using strain in experiments. Table 1 shows representative genes that were expressed in the yeast strains and the genetic origin of the enzymes that exhibited the best activity. Genes from other sources were also found to be active, but are not represented on Table 1.
Example 2– Yeast strains having the ability to produce Cholesterol Saccharomyces cerevisiae, which does not have the ability to naturally produce cholesterol, were genetically modified to upregulate the mevalonate pathway by overexpressing S. cerevisiae tHMG1 driven by a pGAL1 promoter. Additionally, S. cerevisiae were also genetically modified to express two heterologous genes, DHCR7 and DHCR24 driven by a GAL1 or GAL10 promoter. All strains expressed the same DCHR7 from A. thaliana. These different strains were tested for their ability to produce sterol compounds using GC/MS. As shown in FIG. 5, yeast strains expressing a DHCR24, were capable of making cholesterol, where DHCR24 from Homo sapiens and Danio rerio (zebrafish) had the best activity. The yeast strains that did not have a DHCR24 gene, did not produce any cholesterol. Example 3– Converting cholesterol to 7-alpha-hydroxycholesterol S. cerevisiae expressing A. thaliana DHCR7 and H. sapiens DHCR24 were transformed with several variants of cytochrome p450 family 7 subfamily A member 1 (CYP7A1) in combination with different adrenodoxin (ADX) variants. All strains expressed Bos Taurus adrenodoxin reductases (ADRs). The strains were then tested for their ability to convert cholesterol to 7-alpha-hydroxycholesterol, by its ability to hydroxylate the C7 carbon in cholesterol molecules. This conversion was detected by GC/MS. As shown in FIG. 6, CYP7A1 from Mus musculus exhibited the best activity. Activity was also seen in CYP7A1 from Homo sapiens, Rattus norvegicus, Oryctolagus cuniculus, Bos taurus, and Danio rerio. Example 4– Converting 7-alpha-hydroxycholesterol to 7a-hydroxy-4-cholesten-3-one Strains expressing A. thaliana DHCR7 and H. sapiens DHCR24 were genetically engineered to further express M. musculus CYP7A1, ADX from B. taurus and D. rerio, B. Taurus adrenodoxin reductase (ADR), and 3 beta-hydroxysteroid dehydrogenase type 7 (HSD3B7). The strains were then tested by GC/MS for their ability to convert 7-alpha-hydroxycholesterol to 7a-hydroxy-4-cholesten-3-one. As shown in FIG. 7, HSD3B7 from Homo sapiens exhibited the best activity. Activity was also seen in HSD3B7 from Mus musculus and Danio rerio. Example 5– Converting 7a-hydroxy-4-cholesten-3-one to 7a-hydroxy-5b-cholestan-3-one Strains expressing A. thaliana DHCR7 and H. sapiens DHCR24 were genetically engineered to further express M. musculus CYP7A1, ADX from D. rerio and B. taurus, B. taurus ADR, H. sapiens HSD3B7, and aldo-keto reductase family 1 member D1 (AKR1D1). The strains were then tested by GC/MS for their ability to convert 7a-hydroxy-4-cholesten-3-one to 7a-hydroxy-5b-cholestan-3-one. As shown in FIG.8, AKR1D1 from Homo sapiens and Mus musculus exhibited the best activity. Example 6– Converting 7a-hydroxy-5b-cholestan-3-one to 5b-cholestane-3a,7a-diol Strains expressing A. thaliana DHCR7 and H. sapiens DHCR24 were genetically engineered to further express M. musculus CYP7A1, ADX from D. rerio and B. taurus, B. taurus ADR, H. sapiens HSD3B7, M. musculus AKR1D1, and aldo-keto reductase family 1 member C9 (AKR1C9) or aldo- keto reductase family 1 member C4 (AKR1C4). The strains were then tested by GC/MS for their ability to convert 7a-hydroxy-5b-cholestan-3- one to 5b-cholestane-3a,7a-diol. As shown in FIG. 9, AKR1C4 from Macaca fuscata exhibited the best activity. Additionally, AKR1C4 from Homo sapiens exhibited very good activity. Example 7– Converting 7a-hydroxy-4-cholesten-3-one to 7a,12a-dihydroxy-4-cholesten-3-one Strains expressing A. thaliana DHCR7 and H. sapiens DHCR24 were genetically engineered to further express M. musculus CYP7A1, ADX from D. rerio and B. taurus, B. taurus ADR, H. sapiens HSD3B7, and CYP8B1. The strains were then tested by GC/MS for their ability to add a third hydroxyl group to the C12 in the cholesterol backbone. The strains were tested for their ability to produce 7a,12a-dihydroxy- 4-cholesten-3-one from 7a-hydroxy-4-cholesten-3-one. As shown in FIG. 10, CYP8B1 from Mus musculus and Oryctolagus cuniculus exhibited the best activity. CYP8B1 from Homo sapiens and Sus scrofa also exhibited activity. Example 8– Converting 5b-cholestane-3a,7a-diol to (25R)-3a,7a-dihydroxy-5b-cholestanoic acid (and further to (25R)-3a,7a-dihydroxy-5b-cholestanoyl-CoA by coupling with SLC27A5) Strains expressing A. thaliana DHCR7 and H. sapiens DHCR24 and also transformed with other enzymes necessary to produce 5b-cholestane-3a,7a-diol were further genetically engineered to further express different CYP27A1 variants. 7 variants of CYP27A1 were tested in combination with 2 variants of ADX (D. rerio and B. Taurus) and B. Taurus ADR. Additionally, H. sapiens SLC27A5 was expressed to couple this CYP27A1 activity, allowing for detection of the SLC27A5 product by LC-MS instead. As shown in FIG.11, most of the CYP27A1 variants were able to produce the SLC27A5 product. Example 9– Converting (25R)-3a,7a,12a-trihydroxy-5b-cholestan-26-oic acid to (25R)-3a,7a,12a- trihydroxy-5b-cholestanoyl-CoA Variants of solute carrier family 27 member 5 (SLC27A5) were integrated into wild type yeast strains that had been knocked out for the native yeast CoA-ligase, FAT1. The yeast strains were lysed and CoA ligase activity was detected on (25R)-3a,7a,12a-trihydroxy-5b-cholestan-26-oic acid when expressing different variants of SLC27A5. As shown in FIG.12A, HPLC data shows that there is a peak detected which is specific to ligase expressing strains. Further, as shown in FIG. 12B, mass spec data confirms that there exists a peak that confirms the presence of active ligase in the expressing strains. Additionally, CoA ligase also exhibits activity using 3a,5b,7a,12a,24E-trihydroxy-cholest-24-en-26-oic acid as the substrate. Example 10– Converting (25R)-3a,7a-dihydroxy-5b-cholestanoyl-CoA to (25S)-3a,7a-dihydroxy-5b- cholestanoyl-CoA Strains expressing A. thaliana DHCR7, H. sapiens DHCR24, M. musculus CYP7A1, ADX from D. rerio and B. taurus, B. taurus ADR, H. sapiens HSD3B7, M. musculus AKR1D1, M. fuscata AKR1C4, R. norvegicus CYP27A1, H. sapiens SLC27A5, and ACOX2 (from H. sapiens or Oryctolagus cuniculus), were used as background strains to test activity of several alpha-methylacyl-CoA racemases (AMACR). The yeast strains were lysed and (24E)-3a,7a-dihydroxy-5b-cholest-24-enoyl-CoA (product of ACOX2) was measured by LC/MS, since the racemization of (25R)-3a,7a-dihydroxy- 5b-cholestanoyl-CoA to (25S)-3a,7a-dihydroxy-5b-cholestanoyl-CoA is difficult to detect. As shown in FIG.13A, AMACR from both Homo sapiens and Rattus norvegicus produced excellent racemization activity. Further, as shown in FIG.13B, ACOX2 from Homo sapiens in combination with Homo sapien AMACR produces the most (24E)-3a,7a-dihydroxy-5b-cholest-24-enoyl-CoA. Example 11– Converting (25S)-3a,7a-dihydroxy-5b-cholestanoyl-CoA to (24E)-3a,7a-dihydroxy-5b-cholest- 24-enoyl-CoA Strains expressing A. thaliana DHCR7, H. sapiens DHCR24, M. musculus CYP7A1, ADX from D. rerio and B. taurus, B. taurus ADR, H. sapiens HSD3B7, M. musculus AKR1D1, M. fuscata AKR1C4, R. norvegicus CYP27A1, and H. sapiens SLC27A5, and AMACR (from Homo sapiens and Rattus norvegicus), were used as background strains to test activity of different acyl-CoA oxidase 2 (ACOX2). The yeast strains were lysed and (24E)-3a,7a-dihydroxy-5b-cholest-24-enoyl-CoA measured by LC/MS. As shown in FIG.14, ACOX2 from both Homo sapiens and Oryctolagus cuniculus produced the best activity. ACOX2 from Rattus norvegicus, Mus musculus, and Saccharomyces cerevisiae exhibited activity. Example 12– Converting (24E)-3a,7a,12a-trihydroxy-5b-cholest-24-enoyl-CoA to 3a,7a,12a-trihydroxy- 24-oxo-5b-cholestanoyl-CoA Strains expressing SLC27A5-CoA ligases were used as background strains to test activity of different hydroxysteroid 17-beta dehydrogenase 4 (HSD17B4). The yeast strains were lysed and in vitro assays conducted with added substrate 3a,5b,7a,12a,24E-trihydroxy-cholest-24-en-26-oic acid (SLC27A5 CoA-ligase activity has been verified on this substrate). The intermediate product of this bifunctional enzyme HSD17B4, an alcohol, was detected. As shown in FIG.15, HSD17B4 from Rattus norvegicus, Bos taurus, and Xenopus laevis produced the best activity. HSD17B4 from remaining 6 sources also exhibited activity. Example 13– Converting 3a,7a-dihydroxy-24-oxo-5b-cholestanoyl-CoA to 3a,7a-dihydroxy-5b-cholan-24-oyl- CoA Strains expressing A. thaliana DHCR7, H. sapiens DHCR24, M. musculus CYP7A1, ADX from D. rerio and B. taurus, B. taurus ADR, H. sapiens HSD3B7, M. musculus AKR1D1, M. fuscata AKR1C4, R. norvegicus CYP27A1, and H. sapiens SLC27A5, R. norvegicus AMACR, H. sapiens ACOX2, and R. norvegicus HSD17B4 were used as background strains to test activity of sterol carrier protein 2 (SCP2). The background strain was also knocked out for its native yeast gene POT1 which encodes for a 3-ketoacyl-CoA thiolase and expressed Bacteroides fragilis 7a-HSD and Clostridium sardiniense 7b-HSD. Yeast pellets were extracted and subsequently analyzed for relative amounts of UDCA/UDC-CoA product by LC/MS. As shown in FIG. 16, SCP2 activity was detected by LCMS in all samples, including negative control, however enhanced activity was observed in the strain overexpressing the native yeast gene POT1. Example 14– Converting 3a,7a-dihydroxy-5b-cholan-24-oyl-CoA to 3a-hydroxy-7-oxo-5b-cholan-24-oyl-CoA to 3a,7b-dihydroxy-5b-cholan-24-oyl-CoA Strains expressing S. cerevisiae truncated HMG, A. thaliana DHCR7, H. sapiens DHCR24, M. musculus CYP7A1, ADX from D. rerio and B. taurus, B. taurus ADR, H. sapiens HSD3B7, M. musculus AKR1D1, M. fuscata AKR1C4, R. norvegicus CYP27A1, and H. sapiens SLC27A5, R. norvegicus AMACR, H. sapiens ACOX2, and R. norvegicus HSD17B4, S. cerevisiae SCP2, pot1∆, pox1∆, and fox2∆ were used as background strains to determine the working 7alpha and 7beta - hydroxysteroid dehydrogenases, 7a-HSD and 7b-HSD, respectively. Four variants of 7a-HSD (Escherichia coli (strain K12), Luminiphilus syltensis NOR5-1B, Bacteroides fragilis, and Comamonas testosteroni (Pseudomonas testosterone)) were tested in the background strain (in this case also expressing an active C. sardiniense 7b-HSD) for their ability to produce UDC-CoA (also known as 3a,7b-dihydroxy-5b-cholanoyl-CoA having a chemical formula of C45H74N7O19P3S with a mass of 1141.40 and a molecular weight of 1142.10). Cell pellets were collected from 25mL whole cell broth in 24 deep well plates. The cell pellets were re-suspended in a 2 mL 80% Methanol/Water mixture solution, vortexed for 30 minutes at 4°C, centrifuged for 5 minutes at 4°C at 4000 rpm, and transferred 1.8 mL Supernatant to 24 deep well plate. The resulting pellets were dried and re-suspended in 200 µL of a 4:1 MPA (10 mM ammonium formate in water, pH 6):Methanol solution. This resuspension was filtered through a 0.2 µm filter. This final filtered product was measured by liquid chromatography followed by mass spectrometry for the presence of UDC-CoA. A flow chart showing these steps is shown in FIG.3. As shown in FIG.17, 7a-HSD from E. coli and B. fragilis, exhibited significant activity. 7a-HSD from L. syltensis and C. testosterioni showed activity as well. Four variants of 7b-HSD (Pseudomonas syringae pv. atrofaciens, Pseudomonas caricapapayae, Drosophila persimilis (Fruit fly), and Clostridium sardiniense)) were also tested in a background strain (in this case also expressing an active B. fragilis 7a-HSD) for their ability to produce UDC-CoA. The same procedure described above was used. As shown in FIG. 18, 7b-HSD from Clostridium sardiniense exhibited the best activity. 7b-HSD from Pseudomonas caricapapayae also exhibited some activity. Example 15– Confirmation that UDC-CoA was made In order to verify that UDC-CoA from Example 14 was indeed produced, two additional methods of processing samples for use in mass spectrometry were conducted. As seen in FIG.4, the initial pellets were split into two samples. The first sample was washed with 2mL of 80% Methanol/H2O, vortexed, centrifuged, transferred and dried. The first sample, as with the second sample, went through the same processing from this point on. 750 µL of 1N NaOH were added to the pellets and incubated for 60 minutes at 60°C. The sample was then acidified with 500 µL of 2N HCl. 4mL of EtOAc was added and vortexed for 20 minutes. 3mL of the organic layer was removed and dried. This was resuspended in 200 µL methanol and filtered through a 0.45 µM filter. Both direct hydrolysis of the pellets and the indirect hydrolysis of the steroidal-CoA extracts resulted in the detectable UDCA, CDCA, (24E)-3a,7a-dihydroxy-cholest-24-enoic acid, and 3a,7a-dihydroxy-5b-cholestanoic acid. Direct hydrolysis of the pellets seems to yield more. Example 16– Combination of Thiolase/7a-HSD/7b-HSD Strains expressing S. cerevisiae truncated HMG, A. thaliana DHCR7, H. sapiens DHCR24, M. musculus CYP7A1, H. sapiens HSD3B7, M. musculus AKR1D1, M. fuscata AKR1C4, R. norvegicus CYP27A1, and H. sapiens SLC27A5, R. norvegicus AMACR, H. sapiens ACOX2, and R. norvegicus HSD17B4, pot1∆, pox1∆, and fox2∆, were used as background strains to determine the best combination of thiolase/SCP2, 7a-HSD, and 7b-HSD. The strains were then tested by GC/MS for its ability to produce UDCA/UDC-CoA. As seen in FIG.19, the combination of S. cerevisiae POT1 Thiolase, E. coli 7a-HSD, and C. sardiniense 7b-HSD and S. cerevisiae POT1 Thiolase, B. fragilis 7a-HSD, and C. sardiniense 7b-HSD lead to the greatest amounts of UDCA/UDC-CoA production. Other combinations produced detectable levels of UDCA/UDC-CoA production, as seen in FIG.19. Example 17– Identification of Enzymes that Convert Sugar to Cholic Acid and Generating Strains That Can Make Cholic Acid Eleven heterologous enzymes (from the perspective of a Saccharomyces cerevisiae) were identified as possible enzymes that could be used to make cholic acid from cholesterol. See e.g., FIG.22. Two (2) additional enzymes were also identified as possible enzymes that could be used to convert sugar to cholesterol. See e.g., FIG.2. Genes encoding these enzymes were synthesized and then cloned into yeast expression vectors suitable for integration into the yeast genome. These integration constructs were subsequently transformed into Saccharomyces cerevisiae using standard yeast chemical transformation protocol, utilizing Lithium Acetate and PEG (3350). The transformed yeast were grown to mid log phase, then centrifuged at 4000 rpm with the supernatant removed. Pellets were washed with water and centrifuged again. The resulting pellet was resuspended in master mix containing 100 mM lithium acetate, 40% PEG (MW 3,350), 0.35 mg/ml carrier DNA (sheared salmon sperm DNA), and 50 to 500 ng of DNA to be transformed. The cell suspension was then incubated at 30˚C for 30 minutes, followed by at 45 minute heat shock at 42˚C. At this point, nutritional selection was plated, while antifungal selection underwent a 4 hr to overnight recovery in rich yeast media before plating on agar containing the antifungal drug. Plates were then incubated at 30˚C for 2 to 3 days. After colonies were formed, proper integrations were verified by colony PCR before using strain in experiments. Table 2 shows representative genes that were expressed in the yeast strains and the genetic origin of the enzymes that exhibited the best activity. Genes from other sources were also found to be active, but are not represented on Table 2.
Strains with the ability to produce cholesterol were genetically engineered to further express CYP7A1, ADX (2 variants), ADR, and HSD3B7. The activities of CYP7A1 and HSD3B7 were demonstrated as described in Examples 3 and 4. Example 18– Converting 7a-hydroxy-4-cholesten-3-one to 7a,12a-dihydroxy-4-cholesten-3-one Strains expressing A. thaliana DHCR7, H. sapiens DHCR24 were genetically engineered to further express M. musculus CYP7A1, ADX (from D. rerio and B. taurus), B. taurus ADR, H. sapiens HSD3B7, and CYP8B1. The strains were tested for their abilities to produce 7a,12a-dihydroxy-4-cholesten-3-one from 7a-hydroxy-4-cholesten-3-one. As shown in FIG. 23, CYP8B1 from Mus musculus and Oryctolagus cuniculus exhibited the best activity. CYP8B1 from Homo sapiens and Sus scrofa also exhibited activity. Example 19– Confirmation That Choloyl-CoA Was Made Strains expressing S. cerevisiae truncated HMG, A. thaliana DHCR7, H. sapiens DHCR24, M. musculus CYP7A1, B. taurus ADX, B. taurus ADR, H. sapiens HSD3B7, M. musculus AKR1D1, M. fuscata AKR1C4, R. norvegicus CYP27A1, and H. sapiens SLC27A5, R. norvegicus AMACR, H. sapiens ACOX2, R. norvegicus HSD17B4, and S. cerevisiae SCP2 were used as background strains to determine the working CYP8B1. One variant of CYP8B1 was tested (Mus musculus) in the background strain for its ability to produce choloyl-CoA (also known as 3a,7a,12a-trihydroxy-5b-cholan-24-oyl-CoA, having a chemical formula of C 45 H 74 N 7 O 20 P 3 S with a mass of 1157.4 and a molecular weight of 1158.1). The hydrolyzed acid form of choloyl-CoA, cholic acid (also known as 3a,7a,12a-trihydroxy-5b- cholan-24-oic acid, having a chemical formula of C24H40O5 with a mass of 408.3 and a molecular weight of 408.58) was the measureable product. Cell pellets were collected from 15 mL whole cell broth in 24 deep well plates. The cell pellets were re-suspended in a 2 mL 80% Methanol/Water mixture solution, vortexed for 30 minutes at 4 °C, centrifuged for 5 minutes at 4 °C at 4000 rpm, and 1.8 mL supernatant was transferred to 24 deep well plate. The supernatant was dried overnight at 40 °C on centrivap. The dried extracts were hydrolyzed with 750 µL 1N NaOH at 60 °C for 1 hour with vortexing, followed by acidification with 500 µL 2N HCl. The acidified samples were extracted with 4 mL ethyl acetate. 3.5 mL of the organic layer was transferred to a 24 deep well plate and dried at 45 °C on centrivap. The dried extracts were resuspended in 200 µL methanol and filtered through a 0.2 µm filter. This final filtered product was measured by liquid chromatography followed by mass spectrometry for the presence of cholic acid (hydrolyzed choloyl-CoA). A flow chart showing these steps is shown in FIG.24. As shown in FIG. 25, the CYP8B1 from Mus musculus was active and produced choloyl-CoA (cholic acid detected). No cholic acid was detected in the strain lacking the CYP8B1 enzyme.
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