Cells and Methods for the Production of Ursodeoxycholic Acid and Precursors Thereof

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; 7α-hydroxy-4-cholesten-3-one; 7α-hydroxy-5β-cholestan-3-one; 5β-cholestane-3α,7α-diol; (25R)-3α,7α-dihydroxy-5β-cholestanoic acid; (25R)-3α,7α-dihydroxy-5β-chole stanoyl-CoA; (25S)-3α,7α-dihydroxy-5β-cholestanoyl-CoA; (24E) -3α,7α-dihydroxy-5β-cholest-24-enoyl-CoA; 3α,7α-dihydroxy-24-oxo-5β-cholestanoyl-CoA; 3α,7α-dihydroxy-5β-cholan-24-oyl-CoA; 3α-hydroxy-7-oxo-5β-cholan-24-oyl-CoA; 3α,7β-dihydroxy-5β-cholan-24-oyl-CoA; 7α,12α-dihydroxy-4-cholesten-3-one; 7α,12α-dihydroxy-5β-cholestan-3-one; 5β-cholestane-3α,7α,12α-triol; (25R)-3α,7α,12α-trihydroxy-5β-cholestan-26-oic acid; (25R)-3α,7α,12α-trihydroxy-5β-cholestanoyl-CoA; (25S)-3α,7α,12α-trihydroxy-5β-cholestanoyl-CoA; (24E)-3α,7α,12α-trihydroxy-5β-cholest-24-enoyl-CoA; 3α,7α,12α-trihydroxy-24-oxo-5β-cholestanoyl-CoA; 3α,7α,12α-trihydroxy-5β-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, PDX1, HSD17B4, FOX2, SCP2, POT1, ERG10, 7α-HSD, 7β-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 claim 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;

PDX1 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:

7α-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;

7β-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, PDX1, HSD17B4, FOX2, SCP2, POT1, ERG10, 7α-HSD, 7β-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;

PDX1 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:

7α-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;

7β-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, PDX1, HSD17B4, FOX2, SCP2, POT1, ERG10, 7α-HSD, 7β-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;

PDX1 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:

7α-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;

7β-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 Saccharomyces cerevisiae 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 .