5-beta stereoselective reductions of 3-ketochol-4-enoic acid and derivatives thereof

Abstract

Methods of making steroids, methods of making 5β stereoselective reductions of steroids; and steroids produced by said 5β stereoselective reduction.

Description

[0001] 5-BETA STEREOSELECTIVE REDUCTIONS OF 3-KETOCHOL-4- ENOIC ACID AND DERIVATIVES THEREOF

[0002] PRIOR APPLICATIONS

[0003] The present application claims priority to United States Provisional Application Nos. 63 / 537,584 (filed September 11, 2023) and 63 / 643,919 (filed May 8, 2024). The content of the foregoing applications is incorporated by reference as if fully set forth herein.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates generally to methods of making steroids, and to enzymatic 5p stereoselective reductions of 3 -keto-4, 5 -unsaturated steroids to produce the corresponding 5 products.

[0006] REFERENCE TO SEQUENCE LISTING

[0007] The official copy of the Sequence Listing is submitted concurrently with the specification via USPTO Patent Center. This Sequence Listing is part of the specification and is incorporated in its entirety by reference herein. The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on 10 September 2024, is named SHP020.XML and is 62,095 bytes in size.

[0008] BACKGROUND OF THE INVENTION

[0009] Cholic acid and its derivatives find utility in numerous medical applications and research initiatives. Cholic acid itself, sold under the brand name Cholbam®, is approved for use as a treatment for children and adults with bile acid synthesis disorders due to single enzyme defects, and for peroxisomal disorders (such as Zellweger syndrome). 7-Ketolithocholic acid has been examined for its effect on endogenous bile acid synthesis, biliary cholesterol saturation, and its possible role as a precursor of chenodeoxy cholic acid and ursodeoxycholic acid. See Salen et al. Gasteroenterology, 1982;83:341-7. Ursodeoxycholic acid (a / k / a UDCA or ursodiol), sold under the brand name URSO 250® and URSO Forte® tablets, is approved for the treatment of patients with primary biliary cirrhosis (PBC). More recently, obeticholic acid, sold under the brand name Ocaliva®, was approved for the treatment of PBC in combination with UDCA in adults with an inadequate response to UDCA, or as monotherapy in adults unable to tolerate UDCA.

[0010] In spite of this significant medical interest in cholic acid derivatives, methods of synthesizing the derivatives remain cumbersome and inefficient, with numerous processes being proposed. Fantin et al. Steroids, 1993 Nov.; 58:524-526, discloses the preparation of 7a-, 12a-, 12P-hydroxy and 7a-, 12a- and 7a-,12P-dihydroxy-3-ketocholanoic acids by protecting the 3- keto group as dimethyl ketal and subsequent reduction with sodium borohydride of the corresponding 7- and 12-oxo functionalities. WO 2017 / 079062 Al by Galvin reports a method of preparing obeticholic acid by direct alkylation at the C-6 position of 7-keto lithocholic acid (KLCA). WO 2018 / 036982 Al by Bakonyi reports a method of preparing UDCA by oxidizing the 3a- and 7a-hydroxyls on chenodeoxycholic acid (“CDCA”) to 3-keto and 7-keto groups to form diketocholanic acid (“DKCA”), and selectively reducing the 3-keto and 7-keto groups on DKCA to 3a-hydroxyl and 7P-hydroxyl to form UDCA. WO 2021 / 176066 Al by Staunig reports a method of making UDCA by selectively hydroxylating the 7-position on lithocholic acid (“LCA”). He et al., Steroids, 2018 Dec;140: 173-178, discloses a synthetic route of producing ursodeoxycholic acid (UDCA) and obeticholic acid (OCA) through multiple reactions from cheap and readily-available cholic acid.

[0011] Plant sterols as starting materials present a particular challenge because they are initially degraded to intermediates that contain a 3-ketone with a 4,5-double bond that needs to be reduced to give a saturated product possessing 5-beta stereochemistry. CN112375117 (Hunan Kerey Pharmaceutical Co ltd) discloses a multi-step method of synthesizing lithocholic acid from bisnoralcohol through reactions such as side chain construction, hydrogenation, reduction, hydrolysis and the like. Wang et al., Steroids 157 (2020) 108600, report a synthetic route of producing ursodeoxycholic acid (UDCA) through multiple synthetic chemical reactions from commercially available bisnoralcohol. Both produced significant amounts of 5-alpha impurities upon reducing the 4,5-double bond. A. Fauve and A. Kergomard, in Tetrahedron Vol 37. pp. 899 to 901 (1981), report 5-beta stereoselective enzymatic reduction of 4,5 -unsaturated 3-keto- steroids, but the reduction is ineffective (0% yield) when reducing cholestenone, a steroid with a longer side chain. What is needed are more efficient processes for making cholic acid derivatives with high stereoselectivity. Particularly needed are more efficient processes for making 5p-cholic and cholanic acids, including ursodeoxycholic acid, tauroursodeoxycholic acid, and starting materials and intermediates therefor.

[0012] SUMMARY OF INVENTION

[0013] The inventors have discovered novel methods for enzymatically and catalytically reducing the 4,5-double bond of 3-keto-chol-4-enoic acids having 5-carbon side chains at the 17-position to preferentially give 50 products. The methods find particular utility for reducing KCEA and related compounds, for the ultimate production of UDCA and TUDCA, as recited in the first through fifth principal embodiments of this document (hereafter the first set of principal embodiments).

[0014] Thus, in a first principal embodiment the invention provides a method of reducing a 4,5- double bond on 3-ketochol-4-enoic acid (KCEA) or a derivative thereof defined by Formula I, to preferentially give a 5P-product, comprising contacting the compound of Formula I:

[0015] I with At5pStR or an analog thereof having greater than 70%, 80%, 85%, 90%, 95%, or 98% sequence identity with At5pStR, in a reaction mixture under conditions suitable for reducing the 4,5-double bond, thereby producing the compound of Formula II: wherein: (a) A and B are OH and H respectively, H and OH respectively, H and H, or 7-oxo in combination; (b) X is C(O)OR' or C(O)NR1R2; and (c) R1and R2are independently hydrogen, a counterion when the compound is a carboxylate or amide salt, optionally substituted C1-20 alkyl, or optionally substituted aryl. In a second principal embodiment, the invention provides a method of reducing a 4,5- double bond on 3-ketochol-4-enoic acid (KCEA) or a derivative thereof defined by Formula la, to preferentially give a 5 -product, comprising contacting the compound of Formula la: with At5pStR or an analog thereof having greater than 70%, 80%, 85%, 90%, 95%, or 98% sequence identity with At50StR, in a reaction mixture under conditions suitable for reducing the 4,5-double bond, thereby producing the compound of Formula Ila: wherein, in Formulas la and Ila: (a) A and B are OH and H respectively, H and OH respectively, H and H, or 7-oxo in combination; (b) X is C(O)OR1or C(O)NR1R2; (c) Y is H or C(O)OR'; (d) R1and R2are independently hydrogen, a counterion when the compound is a carboxylate or amide salt, optionally substituted C1-20 alkyl, or optionally substituted aryl; and (e) the C22-C23 bond is a single or double bond.

[0016] In a third principal embodiment, the invention provides a method of reducing a 4,5-double bond on 3-ketochol-4-enoic acid (KCEA) or a derivative thereof defined by Formula la, to preferentially give a 5p-product, comprising pumping a solution comprising the compound of Formula la: and an organic solvent through a trickle bed reactor packed with a hydrogenation catalyst, and pumping hydrogen through the trickle bed reactor counter to or concurrently with the solution, thereby producing the compound of Formula Ila:

[0017] Ila wherein, in Formulas la and Ila: (a) A and B are OH and H respectively, H and OH respectively, H and H, or 7-oxo in combination; (b) X is C(O)OR1or C(O)NR1R2; (c) Y is H or C^OR1; (d) R1and R2are independently hydrogen, a counterion when the compound is a carboxylate or amide salt, optionally substituted C1.20 alkyl, or optionally substituted aryl; and (e) the C22-C23 bond is a single or double bond.

[0018] In a fourth principal embodiment the invention provides a method of making UDCA or TUDCA comprising: (a) reducing the 4,5-double bond of a compound of Formula la: to produce the compound of Formula Ila: wherein, in Formulas la and Ila, A and B are OH and H respectively, H and H, or 7-oxo in combination; X is C(O)OR1or C(O)NR'R2; Y is H or C(O)OR1; R1and R2are independently hydrogen, a counterion when the compound is a carboxylate or amide salt, optionally substituted C1-20 alkyl, or optionally substituted aryl; and the C22-C23 bond is a single or double bond; (b) when A and B are H, hydroxylating the 7-H to 7p-hydroxy; (c) reducing the 3-oxo to 3a-hydroxy; (d) when A and B are 7-oxo in combination, reducing the 7-oxo to 7P-hydroxy; (e) when the C22- 23 bond is a double bond, reducing it to a single bond; (f) when Y is C(O)OR1and R1is H, decarboxylating C23 to convert Y to H; (g) when Y is C(O)OR1and R1is optionally substituted C1-20 alkyl or optionally substituted aryl, hydrolyzing Y to convert R1to H and decarboxylating C23 to convert Y to H; (h) when X is a C(O)OR' ester, hydrolyzing the ester to C(O)OH; and (i) when X is a C(O)NR1R2amide and NRJR2is not taurine, hydrolyzing the amide to C(O)OH; thereby producing UDCA or TUDCA, wherein the foregoing steps may be performed simultaneously, sequentially, or in any order.

[0019] In a fifth principal embodiment the invention provides a method of making TUDCA comprising: (a) reducing the 4,5-double bond of a compound of Formula la: o produce the compound of Formula Ila:

[0020] Ila wherein, in Formula la and Ila, A and B are OH and H respectively, H and H, or 7-oxo in combination; X is C(O)OR1or C(O)NR'R2; Y is H or C(O)OR1; R1and R2are independently hydrogen, a counterion when the compound is a carboxylate or amide salt, optionally substituted Ci-20 alkyl, or optionally substituted aryl; and the C22-C23 bond is a single or double bond; (b) when A and B are H, hydroxylating the 7-H to 7p-hydroxy; (c) reducing the 3-oxo to 3a-hydroxy; (d) when A and B are 7-oxo in combination, reducing the 7-oxo to 7P-hydroxy; (e) when the C22- 23 bond is a double bond, reducing it to a single bond; (f) when Y is C(O)OR1and R1is H, decarboxylating C23 to convert Y to H; (g) when Y is C(O)OR1and R1is optionally substituted Ci-20 alkyl or optionally substituted aryl, hydrolyzing Y to convert R1to H and decarboxylating C23 to convert Y to H; (h) when X is a C(O)OR' ester, hydrolyzing the ester to C(O)OH; and (i) when X is a C(O)NR1R2amide, hydrolyzing the amide to C(O)OH; thereby producing UDCA, further comprising: (j) contacting the 24-carboxylic acid with a reagent that converts the 24- carboxylic acid to a derivative that can act as an acylating agent; and (k) reacting the derivative with taurine; thereby forming TUDCA, wherein steps (a) through (i) may be performed simultaneously, sequentially, or in any order. The methods also can be used to reduce the 4,5-double bond of bisnoralcohol and related compounds to preferentially give 5p products, defined by the sixth through eighth principal embodiments (hereafter the second set of principal embodiments).

[0021] Thus, a sixth principal embodiment of the invention provides a method of reducing a 4,5- double bond on (20S)-21-hydroxy-20-methylpregn-4-en-3-one (bisnoralcohol, or BA) or a derivative thereof defined by Formula III, to preferentially give a 5P-product, comprising contacting the compound of Formula III: with At5pStR or an analog thereof having greater than 70%, 80%, 85%, 90%, 95%, or 98% sequence identity with At5pStR, in a reaction mixture under conditions suitable for reducing the 4,5-double bond, thereby producing the compound of Formula IV: wherein: (a) A and B are OH and H respectively, H and OH respectively, H and H, or 7-oxo in combination; and (b) X’ is hydrogen or a protecting group.

[0022] In a seventh principal embodiment the invention provides a method of making UDCA comprising: (a) reducing the 4,5-double bond of a compound of Formula III: to produce the compound of Formula IV:

[0023] IV wherein, in Formulas III and IV, A and B are OH and H respectively, H and H, or 7-oxo in combination, and X’ is hydrogen or a protecting group; further comprising: (b) when A and B are H, hydroxylating the 7-H to 70-hydroxy; (c) reducing the 3-oxo to 3a-hydroxy; (d) converting the 21 -OX’ group to a 5-carbon cholanic acid side chain in which OX’ is replaced by CH2CO2H; and (e) when A and B are 7-oxo in combination, reducing the 7-oxo to 7p~hydroxy; thereby producing UDCA, wherein the foregoing steps may be performed simultaneously, sequentially, or in any order.

[0024] In an eighth principal embodiment the invention provides a method of making TUDCA comprising: (a) reducing the 4,5-double bond of a compound of Formula III: to produce the compound of Formula IV:

[0025] IV wherein, in Formulas III and IV, A and B are OH and H respectively, H and H, or 7-oxo in combination, and X’ is hydrogen or a protecting group; further comprising: (b) when A and B are H, hydroxylating the 7-H to 7p-hydroxy; (c) reducing the 3-oxo to 3a-hydroxy; (d) converting the 21 -OX’ to a 5-carbon cholanic acid side chain in which OX’ is replaced by CH2CO2H carboxylic acid; (e) when A and B are 7-oxo in combination, reducing the 7-oxo to 7P-hydroxy; (f) contacting the 24-carboxylic acid with a reagent that converts the 24-carboxylic acid to a derivative that can act as an acylating agent; and (g) reacting the derivative with taurine; thereby forming TUDCA, wherein steps (a) through (g) may be performed simultaneously, sequentially, or in any order, except that step (d) must be done before step (f), and step (g) must be performed after step (f).

[0026] A third set of principal embodiments (principal embodiments nine through eleven) relate to the exceptionally pure products obtained from the reduction of the 4,5-double bonds on Formula I, Formula la, and Formula III. Thus, in a ninth principal embodiment the invention provides a compound of Formula II: comprising a 813C value corresponding to a plant derived molecule, preferably comprising less than -15%o, -17.5%o, -20%o, -22.5%o, or -25%o 813C relative to VPDB, and an isomeric purity relative to the corresponding 5a-isomer greater than 95%, 98%, 99%, 99.5%, or 99.8%; wherein:

[0027] (a) A and B are OH and H respectively, H and OH respectively, H and H, or 7-oxo in combination;

[0028] (b) X is C(O)OR1or C(O)NR'R2; (c) R1and R2are independently hydrogen, a counterion when the compound is a carboxylate or amide salt, optionally substituted C1-20 alkyl, or optionally substituted aryl.

[0029] In a tenth principal embodiment the invention provides a compound of Formula Ila: comprising a 813C value corresponding to a plant derived molecule, preferably comprising less than -15%o, -17.5%o, -20%o, -22.5%o, or -25%o 813C relative to VPDB, and an isomeric purity relative to the corresponding 5a-isomer greater than 98%, 99%, 99.5%, 99.7%, 99.8%, 99.9%, 99.95%, 99.97%, or 99.98%; wherein: (a) A and B are OH and H respectively, H and H, or 7-oxo in combination; (b) X is C(O)OR1or C(O)NR1R2; (c) Y is H or C(O)OR1; (d) R1and R2are independently hydrogen, a counterion when the compound is a carboxylate or amide salt, optionally substituted C1-20 alkyl, or optionally substituted aryl; and (e) the C22-C23 bond is a single or double bond. In an eleventh principal embodiment, the invention provides a compound of Formula IV: comprising a 613C value corresponding to a plant derived molecule, preferably comprising less than -15%o, -17.5%o, -20%o, -22.5%o, or -25%o 813C relative to VPDB, and an isomeric purity relative to the corresponding 5a-isomer greater than 98%, 99%, 99.5%, 99.7%, 99.8%, 99.9%, 99.95%, 99.97%, or 99.98%; wherein: (a) A and B are OH and H respectively, H and H, or 7-oxo in combination; and (b) X’ is hydrogen or a protecting group.

[0030] Additional advantages of the invention are set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

[0031] BRIEF DESCRIPTION OF THE FIGURES

[0032] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and together with the description serve to explain the principles of the invention.

[0033] FIGURE l is a graphical depiction of the resulting HPLC-UV traces from the experiment described in Example 6E, using strain E. coli sp. SAND201. A: UV trace of the KCEA standard. B: UV trace of the 3-KCA standard. C: UV trace of the extracted reaction mixture.

[0034] FIGURE 2 is a graphical depiction of the resulting HPLC-UV traces from the experiment described in Example 6E, using strain E. coli sp. SAND203. A: UV trace of the KCEA standard. B: UV trace of the 3-KCA standard. C: UV trace of the extracted reaction mixture.

[0035] FIGURE 3 is a graphical depiction of the resulting HPLC-UV traces from the experiment described in Example 6E, using strain E. coli sp. SAND204. A: UV trace of the KCEA standard. B: UV trace of the 3-KCA standard. C: UV trace of the extracted reaction mixture. FIGURE 4 is a graphical depiction of the resulting HPLC-UV traces from the experiment described in Example 6G, using strain P. pastoris sp. SAND205. A: UV trace of the KCEA standard. B: UV trace of the 3-KCA standard. C: UV trace of the extracted reaction mixture.

[0036] FIGURE 5 is a graphical depiction of the resulting HPLC-UV traces from the experiment described in Example 6H. A: UV trace of the KCEA standard. B: UV trace of the 3-KCA standard. C: UV trace of the extracted whole-cell bioconversion mixture.

[0037] DETAILED DESCRIPTION OF THE INVENTION

[0038] De finitions and Use o f Terms

[0039] As used in the specification and claims, the singular forms a, an, and the include plural references unless the context clearly dictates otherwise. For example, the term “a specification” refers to one or more specifications for use in the presently disclosed methods and systems. “A hydrocarbon” includes mixtures of two or more such hydrocarbons, and the like. The word “or” or like terms as used herein means any one member of a particular list and also includes any combination of members of that list.

[0040] As used in this specification and in the claims which follow, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. When an element is described as comprising one or a plurality of components, steps or conditions, it will be understood that the element can also be described as “consisting of’ or “consisting essentially of’ the component, step or condition, or the plurality of components, steps or conditions.

[0041] When used herein the term “about” will compensate for variability allowed for in the pharmaceutical industry and inherent in pharmaceutical products. In one embodiment the term allows for any variation within 5% of the recited specification or standard. In one embodiment the term allows for any variation within 10% of the recited specification or standard. All numerical values recited in the application should be interpreted as preceded by the term “about,” thereby providing additional embodiments with 5% variation and 10% variation.

[0042] When a method is defined by its constituent steps, it will be understood that the method includes the steps performed simultaneously, sequentially, or in any order, unless specified to the contrary. When a method can be performed simultaneously, sequentially or in any order, and that method is expressed in a dependent claim, it will be understood that the steps of that dependent claim and any claims on which that dependent claim depends can be performed simultaneously, sequentially, or in any order.

[0043] As used herein, the structure - refers to a bond which can be either a single covalent bond or a double bond.

[0044] In the case of Formula I, Formula la, or Formula III when groups A and B in combination form a 7-oxo, it is understood that the 4,5-double bond can reversibly shift to the 5,6-position and that either of the ketones may convert to its enol form to give a conjugated dienol-ketone structure.

[0045] At5pStR refers to the 388 amino acid D4,5-steroid 5b-reductase (At5b-StR) described by V. Herl et al. in Biochimie 91 (2009) 517-525 (“Herl”) (SEQ ID NO: 29). The contents of Herl are incorporated herein in their entirety as if expressly set forth herein, including the cloning, functional expression of a VEP1 cDNA from A. thaliana in Escherichia coli, the biochemical characterization of the respective gene product (AY062451) as a D4,5-steroid 5b-reductase (At5b- StR), and the modelling of the active site of the enzyme.

[0046] 3-KCA, or 3-keto-5p-cholanic acid, has the following chemical structure:

[0047] Bisnoralcohol, (20S)-21-hydroxy-20-methylpregn-4-en-3-one, or BA, has the following chemical structure:

[0048] Ursodeoxycholic acid, 3a,7P-dihydroxy-5P-cholanic acid, or simply ursodiol or UDCA, has the following chemical structure:

[0049] Tauroursodeoxy cholic acid, or TUDCA, has the following chemical structure:

[0050] KCEA, or 3-ketochol-4-enoic acid, has the following chemical structure:

[0051] KUDCA, or 3 -ketoursodeoxy cholic acid, or 3-keto-7p-hydroxy-5p-cholanic acid has the following chemical structure:

[0052] “Naturally-occurring” or “wild-type” or “native,” in contrast to “non-naturally occurring,” “non-wild-type,” “non-native,” or “foreign,” refers to the form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.

[0053] “Recombinant” when used with reference to, e.g., a cell, nucleic acid, or polypeptide, refers to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature. Non-limiting examples include, among others, recombinant cells expressing genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise expressed at a different level.

[0054] “Percentage of sequence identity” and “percentage homology” and like terms are used interchangeably herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window will comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0055] Those of skill in the art will appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, ajoint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, J. Mol. Biol. 215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively.

[0056] “Reference sequence” refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity.

[0057] “Comparison window” refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 contiguous residues, and includes, optionally 30, 40, 50, 100, 150, or 200 or longer windows.

[0058] “Substantial identity” refers to a polynucleotide or polypeptide sequence that has at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90% sequence identity, or at least 95 percent sequence identity, potentially at least 98 or 99% sequence identity as compared to a reference sequence over a comparison window comprising at least 90%, 95%, 98%, or 99% of the reference sequence. In specific embodiments applied to polypeptides, the term “substantial identity” means that two polypeptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, at least 95 percent sequence identity or more (e.g., 99 percent sequence identity). Preferably, residue positions which are not identical differ by conservative amino acid substitutions. When a sequence identity is given for an amino acid or nucleic acid sequence herein, it will be understood that the sequence identity can be replaced by any of the foregoing sequence identities to form separate embodiments.

[0059] When reference to a cellular organism is given herein, it will be understood to refer to the organism in both its wild-type state and as a modified organism. The term Escherichia coli thus includes all of the wild-type man-made bacteria existing naturally in nature, in addition to any man-made bacteria produced using recombinant techniques.

[0060] When purities are given herein based on percentages, they will be understood to be based on weight percent. Likewise, when yields are expressed as percentages, they will be understood to refer to the weight of the resultant product actually observed relative to the weight of the resultant product had there been 100% conversion. “Recovered” as used herein in relation to an enzyme, protein, or compound (e.g., UDCA), refers to a more or less pure form of the enzyme, protein, or compound. In like manner, when an enzyme, protein or compound is recovered from a medium such as a reaction medium, it will be understood to be more or less free of the medium (e.g. containing no more than 95%, 98%, or 99.5% of the other components of the reaction medium).

[0061] When a compound is said herein to be “substantially pure” or “substantially free of’ one or more impurities, it will be understood to comprise at least 98%, 99%, 99.5%, 99.7%, 99.8%, 99.9%, 99.95%, 99.97%, or 99.98% of the compound, or no more than 2%, 1%, 0.5%, 0.3%, 0.2%, 0.1%, 0.05%, 0.03%, or 0.02% of the one or more impurities, unless stated to the contrary, all percentages being weight-based percentages (w / w).

[0062] The terms “purity” and “free of’ are conversely related. Thus, a compound that is “substantially pure” is also “substantially free of impurities,” and a compound that is greater than 98% or 99% pure will contain no more than 2% or 1% impurities, respectively, where impurities are defined as any substances or species other than the reference compound. In like manner, a 5P- isomer having an isomeric purity relative to the corresponding 5a-isomer of greater than 98% or 99% can also be said to be free of no more than 2% or 1% of the corresponding 5a-isomer.

[0063] All purity percentages stated herein are weight-based percentages unless specifically stated to the contrary.

[0064] Discussion

[0065] In a first principal embodiment the invention provides a method of reducing a 4,5-double bond on 3 -ketochol -4-enoic acid (KCEA) or a derivative thereof defined by Formula I, to preferentially give a 5 P -product, comprising contacting the compound of Formula I:

[0066] I with At5pStR or an analog thereof having greater than 70%, 80%, 85%, 90%, 95%, or 98% sequence identity with At5pStR, in a reaction mixture under conditions suitable for reducing the 4,5-double bond, thereby producing the compound of Formula II: wherein: (a) A and B are OH and H respectively, H and OH respectively, H and H, or 7-oxo in combination; (b) X is C(O)OR1or C(O)NR1R2; and (c) R1and R2are independently hydrogen, a counterion when the compound is a carboxylate or amide salt, optionally substituted C1-20 alkyl, or optionally substituted aryl. The first principal embodiment further provides (i) a compound of Formula II, (ii) Compound lb, 3b, 4b, 5b, 6b, 7b, or 9b (as defined elsewhere in this document), and (iii) 3-KCA, obtained by the method of the first principal embodiment.

[0067] In a second principal embodiment the invention provides a method of reducing a 4,5- double bond on 3-ketochol-4-enoic acid (KCEA) or a derivative thereof defined by Formula la, to preferentially give a 50-product, comprising contacting the compound of Formula la: with At50StR or an analog thereof having greater than 70%, 80%, 85%, 90%, 95%, or 98% sequence identity with At50StR, in a reaction mixture under conditions suitable for reducing the 4,5-double bond, thereby producing the compound of Formula Ila: wherein, in Formulas la and Ila: (a) A and B are OH and H respectively, H and OH respectively, H and H, or 7-oxo in combination; (b) X is C(O)OR' or C(O)NR1R2; (c) Y is H or C(O)ORJ; (d) R1and R2are independently hydrogen, a counterion when the compound is a carboxylate or amide salt, optionally substituted C1.20 alkyl, or optionally substituted aryl; and (e) the C22-C23 bond is a single or double bond. The second principal embodiment further provides (i) a compound of Formula Ila, (ii) Compound lb, 3b, 4b, 5b, 6b, 7b, or 9b (as defined elsewhere in this document), and (iii) 3-KCA, obtained by the method of the second principal embodiment.

[0068] In a third principal embodiment, the invention provides a method of reducing a 4,5-double bond on 3-ketochol-4-enoic acid (KCEA) or a derivative thereof defined by Formula la, to preferentially give a 5p-product, comprising pumping a solution comprising the compound of Formula la: and an organic solvent through a trickle bed reactor packed with a hydrogenation catalyst, and pumping hydrogen through the trickle bed reactor counter to or concurrently with the solution, thereby producing the compound of Formula Ila: wherein, in Formulas la and Ila: (a) A and B are OH and H respectively, H and OH respectively, H and H, or 7-oxo in combination; (b) X is C(O)OR' or C(O)NR'R2; (c) Y is H or C(O)OR1; (d) R1and R2are independently hydrogen, a counterion when the compound is a carboxylate or amide salt, optionally substituted C1.20 alkyl, or optionally substituted aryl; and (e) the C22-C23 bond is a single or double bond. The third principal embodiment further provides (i) a compound of Formula Ila, (ii) Compound lb, 3b, 4b, 5b, 6b, 7b, or 9b (as defined elsewhere in this document), and (iii) 3-KCA, obtained by the method of the third principal embodiment.

[0069] In a fourth principal embodiment the invention provides a method of making UDCA or TUDCA comprising: (a) reducing the 4,5-double bond of a compound of Formula la (preferably KCEA): to produce the compound of Formula Ila: wherein, in Formulas la and Ila, A and B are OH and H respectively, H and H, or 7-oxo in combination; X is C(O)OR1or C(O)NR'R2; Y is H or C(O)OR1; R1and R2are independently hydrogen, a counterion when the compound is a carboxylate or amide salt, optionally substituted C1-20 alkyl, or optionally substituted aryl; and the C22-C23 bond is a single or double bond; (b) when A and B are H, hydroxylating the 7-H to 70-hydroxy; (c) reducing the 3-oxo to 3a-hydroxy; (d) when A and B are 7-oxo in combination, reducing the 7-oxo to 7P-hydroxy; (e) when the C22- 23 bond is a double bond, reducing it to a single bond; (f) when Y is C(O)OR1and R1is H, decarboxylating C23 to convert Y to H; (g) when Y is C(O)OR1and R1is optionally substituted C1-20 alkyl or optionally substituted aryl, hydrolyzing Y to convert R1to H and decarboxylating C23 to convert Y to H; (h) when X is a C(O)OR1ester, hydrolyzing the ester to C(O)OH; and (i) when X is a C^lSn R2amide and NR'R2is not taurine, hydrolyzing the amide to C(O)OH; thereby producing UDCA or TUDCA, wherein the foregoing steps may be performed simultaneously, sequentially, or in any order. The fourth principal embodiment further provides UDCA or TUDCA obtained by the method of the fourth principal embodiment.

[0070] In a fifth principal embodiment the invention provides a method of making TUDCA comprising: (a) reducing the 4,5-double bond of a compound of Formula la (preferably KCEA): o produce the compound of Formula Ila: wherein, in Formulas la and Ila, A and B are OH and H respectively, H and H, or 7-oxo in combination; X is C(O)OR1or C(O)NR1R2; Y is H or C(O)OR1; R1and R2are independently hydrogen, a counterion when the compound is a carboxylate or amide salt, optionally substituted Ci-20 alkyl, or optionally substituted aryl; and the C22-C23 bond is a single or double bond; (b) when A and B are H, hydroxylating the 7-H to 7P-hydroxy; (c) reducing the 3-oxo to 3a-hydroxy; (d) when A and B are 7-oxo in combination, reducing the 7-oxo to 7P-hydroxy; (e) when the C22- 23 bond is a double bond, reducing it to a single bond; (f) when Y is C(O)OR1and R1is H, decarboxylating C23 to convert Y to H; (g) when Y is C(O)OR1and R1is optionally substituted Ci-20 alkyl or optionally substituted aryl, hydrolyzing Y to convert R1to H and decarboxylating C23 to convert Y to H; (h) when X is a C(O)OR1ester, hydrolyzing the ester to C(O)OH; and (i) when X is a C(O)NR1R2amide, hydrolyzing the amide to C(O)OH; thereby producing UDCA, further comprising: (j) contacting the 24-carboxylic acid with a reagent that converts the 24- carboxylic acid to a derivative that can act as an acylating agent; and (k) reacting the derivative with taurine; thereby forming TUDCA, wherein steps (a) through (i) may be performed simultaneously, sequentially, or in any order. The fifth principal embodiment further provides TUDCA obtained by the method of the fifth principal embodiment.

[0071] A sixth principal embodiment of the invention provides a method of reducing a 4,5-double bond on (20S)-21-hydroxy-20-methylpregn-4-en-3-one (bisnoralcohol, or BA) or a derivative thereof defined by Formula III, to preferentially give a 5P-product, comprising contacting the compound of Formula III: with At5pStR or an analog thereof having greater than 70%, 80%, 85%, 90%, 95%, or 98% sequence identity with At5 StR, in a reaction mixture under conditions suitable for reducing the 4,5-double bond, thereby producing the compound of Formula IV:

[0072] IV wherein: (a) A and B are OH and H respectively, H and OH respectively, H and H, or 7-oxo in combination; and (b) X’ is hydrogen or a protecting group. The sixth principal embodiment further provides a compound of formula IV, Compound 2b, or Compound 8b (as defined elsewhere in this document) obtained by the method of the sixth principal embodiment.

[0073] A seventh principal embodiment the invention provides a method of making UDCA comprising: (a) reducing the 4,5-double bond of a compound of Formula III (preferably BA): to produce the compound of Formula IV:

[0074] IV wherein, in Formulas III and IV, A and B are OH and H respectively, H and H, or 7-oxo in combination, and X’ is hydrogen or a protecting group; further comprising: (b) when A and B are H, hydroxylating the 7-H to 7p-hydroxy; (c) reducing the 3-oxo to 3a-hydroxy; (d) converting the 21 -OX’ group to a 5-carbon cholanic acid side chain in which OX’ is replaced by CH2CO2H; and (e) when A and B are 7-oxo in combination, reducing the 7-oxo to 7P-hydroxy; thereby producing UDCA, wherein the foregoing steps may be performed simultaneously, sequentially, or in any order. The seventh principal embodiment further provides UDCA obtained by the method of the seventh principal embodiment.

[0075] An eighth principal embodiment of the invention provides a method of making TUDCA comprising: (a) reducing the 4,5-double bond of a compound of Formula III (preferably BA): to produce the compound of Formula IV:

[0076] IV wherein, in Formulas III and IV, A and B are OH and H respectively, H and H, or 7-oxo in combination, and X’ is hydrogen or a protecting group; further comprising: (b) when A and B are H, hydroxylating the 7-H to 7p-hydroxy; (c) reducing the 3-oxo to 3a-hydroxy; (d) converting the 21 -OX’ to a 5-carbon cholanic acid side chain in which OX’ is replaced by CH2CO2H carboxylic acid; (e) when A and B are 7-oxo in combination, reducing the 7-oxo to 7P-hydroxy; (f) contacting the 24-carboxylic acid with a reagent that converts the 24-carboxylic acid to a derivative that can act as an acylating agent; and (g) reacting the derivative with taurine; thereby forming TUDCA, wherein steps (a) through (g) may be performed simultaneously, sequentially, or in any order, except that step (d) must be done before step (f), and step (g) must be performed after step (f). The eighth principal embodiment further provides TUDCA obtained by the method of the eighth principal embodiment.

[0077] In a ninth principal embodiment the invention provides a compound of Formula II (preferably 3-KCA): comprising a 813C value corresponding to a plant derived molecule, preferably comprising less than -15%o, -17.5%o, -20%o, -22.5%o, or -25%o 813C relative to VPDB, and an isomeric purity relative to the corresponding 5a-isomer greater than 95%, 98%, 99%, 99.5%, or 99.8% (w / w); wherein: (a) A and B are OH and H respectively, H and OH respectively, H and H, or 7-oxo in combination; (b) X is C(O)OR' or C(O)NR' R2; and (c) R1and R2are independently hydrogen, a counterion when the compound is a carboxylate or amide salt, optionally substituted C1-20 alkyl, or optionally substituted aryl.

[0078] In an especially preferred aspect of the ninth principal embodiment, the compound of formula II has an isomeric purity relative to the corresponding 5a-isomer of 100%. In an especially preferred aspect of the ninth principal embodiment, the compound of Formula II is selected from a compound of Formula lb, 3b, 4b, 5b, 6b, 7b, or 9b (as defined elsewhere in this document) and has an isomeric purity relative to the corresponding 5a-isomer of 100%. In another especially preferred aspect of the ninth principal embodiment, the compound of formula II is 3-KCA and has an isomeric purity relative to the corresponding 5a-isomer of 100%. In another preferred aspect of the ninth principal embodiment, the compound of the ninth principal embodiment is obtained by the method of the first principal embodiment.

[0079] In a tenth principal embodiment the invention provides a compound of Formula Ila (preferably 3-KCA): comprising a 813C value corresponding to a plant derived molecule, preferably comprising less than -15%o, -17.5%o, -20%o, -22.5%o, or -25%o 613C relative to VPDB, and an isomeric purity relative to the corresponding 5a-isomer greater than 98%, 99%, 99.5%, 99.7%, 99.8%, 99.9%, 99.95%, 99.97%, or 99.98% (w / w); wherein: (a) A and B are OH and H respectively, H and H, or 7-oxo in combination; (b) X is C(O)OR1or C(O)NR1R2; (c) Y is H or C(O)OR1; (d) R1and R2are independently hydrogen, a counterion when the compound is a carboxylate or amide salt, optionally substituted C1-20 alkyl, or optionally substituted aryl; and (e) the C22-C23 bond is a single or double bond.

[0080] In an especially preferred aspect of the tenth principal embodiment, the compound of formula Ila has an isomeric purity relative to the corresponding 5a-isomer of 100%. In an especially preferred aspect of the tenth principal embodiment, the compound of Formula Tla is selected from a compound of Formula lb, 3b, 4b, 5b, 6b, 7b, or 9b (as defined elsewhere in this document) and has an isomeric purity relative to the corresponding 5a-isomer of 100%. In another especially preferred aspect of the tenth principal embodiment, the compound of formula Ila is 3- KCA and has an isomeric purity relative to the corresponding 5a-isomer of 100%. In another preferred aspect of the tenth principal embodiment, the compound of the tenth principal embodiment is obtained by the method of the second principal embodiment.

[0081] In an eleventh principal embodiment, the invention provides a compound of Formula IV: comprising a 513C value corresponding to a plant derived molecule, preferably comprising less than -15%o, -17.5%o, -20%o, -22.5%o, or -25%o 6I 3C relative to VPDB, and an isomeric purity relative to the corresponding 5a-isomer greater than 98%, 99%, 99.5%, 99.7%, 99.8%, 99.9%, 99.95%, 99.97%, or 99.98% (w / w); wherein: (a) A and B are OH and H respectively, H and H, or 7-oxo in combination; and (b) X’ is hydrogen or a protecting group.

[0082] In an especially preferred aspect of the eleventh principal embodiment, the compound of formula IV has an isomeric purity relative to the corresponding 5a-isomer of 100%. In another especially preferred aspect of the eleventh principal embodiment, the compound of Formula IV is selected from a compound of Formula 2b or 8b (as defined elsewhere in this document) and has an isomeric purity relative to the corresponding 5a-isomer of 100%. In another especially preferred aspect of the eleventh principal embodiment, the compound of formula IV is defined when A and B are H, X’ is H, and the compound has an isomeric purity relative to the corresponding 5a-isomer of 100%. In another preferred aspect of the eleventh principal embodiment, the compound of the eleventh principal embodiment is obtained by the method of the sixth principal embodiment.

[0083] When considering KCEA and its derivatives, either as substrates in the first set of principal embodiments, or when considering 3-KCA and its derivatives (i.e. the end-products following the 4,5-double bond reduction of KCEA and its derivatives or the compounds of principal embodiments nine and ten), preferred genre of Formula I or Formula la, and preferred genre of Formula II and Formula Ila, can be defined when: • A and B are 7-oxo in combination;

[0084] • A and B are H;

[0085] • A is H and B is OH;

[0086] • A is OH and B is H;

[0087] • X is CCO R1;

[0088] • X is C(O)OH;

[0089] • A and B are 7-oxo in combination and X is C(O)OR';

[0090] • A and B are H and X is C(O)OR1;

[0091] • A is H and B is OH and X is C(O)OR'; or

[0092] • A is OH and B is H and X is C(O)OR'.

[0093] Additional preferred genre of Formula la and Formula Ila can be defined when:

[0094] • A and B are 7-oxo in combination, X is C(O)OR1, Y is H, and the C22-C23 bond is a single bond;

[0095] • A and B are H, X is C(O)OR1, Y is H, and the C22-C23 bond is a single bond.

[0096] • A and B are OH and H, respectively, X is C(O)OR1, Y is H, and the C22-C23 bond is a single bond;

[0097] • A and B are 7-oxo in combination, X is C(O)OH, Y is H, and the C22-C23 bond is a single bond;

[0098] • A and B are H, X is C(O)OH, Y is H, and the C22-C23 bond is a single bond;

[0099] • A and B are OH and H, respectively, X is C(O)OH, Y is H, and the C22-C23 bond is a single bond.

[0100] A particularly preferred species of Formulas I and la is KCEA. A particularly preferred species of Formulas II and Ila is 3-KCA.

[0101] Any of Formulas I, la, II, and Ila can be further defined when - is a single bond.

[0102] Alternatively, any of Formulas I, la, II, and Ila can be further defined when > - is a double bond.

[0103] In one particular embodiment of Formulas I, la, II, and Ila, A and B are 7-oxo in combination, X is C(O)OR1, and > - is a double bond.

[0104] Preferred KCEA derivatives falling under the definition of Formula I or Formula la can also be defined by the following structures la, 3a, 4a, 5a, 6a, 7a, or 9a, and the resulting products falling under the definition of Formula II or Ila defined by structures lb, 3b, 4b, 5b, 6b, 7b, or 9b, respectively:

[0105] In like manner, when considering BA and its derivatives, either as substrates in the second set of principal embodiments, or when considering the end-products following the 4,5-double bond reduction of BA and its derivatives or the compounds of principal embodiment eleven), preferred genre of Formula III and Formula IV can be defined when:

[0106] • A and B are H;

[0107] • A and B are H and X’ forms with the O to which it is attached an ester, an ether, a silyl ether, or an acetal;

[0108] • A and B are H and X’ is H;

[0109] • A and B are 7-oxo in combination;

[0110] • A and B are 7-oxo in combination and X’ forms with the O to which it is attached an ester, an ether, a silyl ether, or an acetal;

[0111] • A and B are 7-oxo in combination and X’ is H;

[0112] • A and B are OH and H, respectively;

[0113] • A and B are OH and H, respectively, and X’ forms with the O to which it is attached an ester, an ether, a silyl ether, or an acetal; and

[0114] • A and B are OH and H, respectively, and X’ is H.

[0115] Preferred species falling under the definition of Formula III can also be defined by structures 2a and 8a, with the resulting product falling under the definition of Formula IV defined by structure 2b or 8b, respectively:

[0116] In the first, second and third principal embodiments, the methods can further be defined based on the subsequent conversion of the reduced KCEA or KCEA derivative to UDCA or TUDCA. Thus, in another embodiment when A and B are 7-oxo in combination and Y is H when present, the method further comprises: (a) reducing the 3-oxo to 3a-hydroxy; (b) reducing the 7- oxo to 7p-hydroxy; (c) when the C22-C23 bond is a double bond, reducing it to a single bond; (d) when X is a C(O)OR1ester, hydrolyzing the ester; and (e) when X is a C(O)NR1R2amide and NR'R2is not taurine, hydrolyzing the amide; thereby producing UDCA, wherein the foregoing steps may be performed simultaneously, sequentially, or in any order. In another embodiment when UDCA is formed, the method further comprises (a) contacting the 24-carboxylic acid on UDCA with a reagent that converts the acid to a derivative that can act as an acylating agent; and (b) reacting the derivative with taurine to form TUDCA.

[0117] In another embodiment when A and B are H and Y is H when present, the method further comprises: (a) reducing the 3-oxo to 3a-hydroxy; (b) when the C22-C23 bond is a double bond, reducing it to a single bond; (c) hydroxylating the 7-H to 70-hydroxy; (d) when X is a C(O)OR1ester, hydrolyzing the ester; and (e) when X is a C(O)NR1R2amide and NR'R2is not taurine, hydrolyzing the amide to C(O)OH; thereby producing UDCA, wherein the foregoing steps may be performed simultaneously, sequentially, or in any order. In another embodiment when UDCA is formed , the method further comprises (a) contacting the 24-carboxylic acid on UDCA with a reagent that converts the acid to a derivative that can act as an acylating agent; and (b) reacting the derivative with taurine to form TUDCA.

[0118] In another embodiment when A and B are OH and H, respectively, and Y is H when present, the method further comprises: (a) reducing the 3-oxo to 3a-hydroxy; (b) when the C22- C23 bond is a double bond, reducing it to a single bond; (c) when X is a C(O)OR1ester, hydrolyzing the ester; and (d) when X is a C(O)NR1R2amide and NR'R2is not taurine, hydrolyzing the amide; thereby producing UDCA, wherein the foregoing steps may be performed simultaneously, sequentially, or in any order. In another embodiment, when UDCA is formed the method further comprises (a) contacting the 24-carboxylic acid on UDCA with a reagent that converts the acid to a derivative that can act as an acylating agent; and (b) reacting the derivative with taurine to form TUDCA.

[0119] In like manner, the methods of the sixth principal embodiment can further be defined based on the subsequent conversion of the compound of Formula IV to UDCA or TUDCA. This, in one embodiment when A and B are H, the method further comprises: (a) converting the 21-alcohol group to the 5-carbon cholanic acid side chain terminating in 24-carboxylic acid; (b) reducing the 3-oxo to 3a-hydroxy; and (c) hydroxylating the 7-H to 7P-hydroxy, to produce UDCA, wherein the foregoing steps may be performed simultaneously, sequentially, or in any order. In another embodiment, the method further comprises (a) contacting the 24-carboxylic acid on UDCA with a reagent that converts the acid to a derivative that can act as an acylating agent; and (b) reacting the derivative with taurine to form TUDCA.

[0120] In another embodiment, when A and B are OH and H, respectively, the method further comprises: (a) converting the 21-alcohol group to the 5-carbon cholanic acid side chain terminating in 24-carboxylic acid; and (b) reducing the 3-oxo to 3a-hydroxy, to produce UDCA; , wherein the foregoing steps may be performed simultaneously, sequentially, or in any order. In another embodiment, the method further comprises (a) contacting the 24-carboxylic acid on UDCA with a reagent that converts the acid to a derivative that can act as an acylating agent; and (b) reacting the derivative with taurine to form TUDCA.

[0121] In another embodiment, wherein A and B form 7-oxo in combination, the method further comprises: (a) converting the 21-OX’ group to a 5-carbon cholanic acid side chain in which OX’ is replaced by CH2CO2H; (b) reducing the 3-oxo to 3a-hydroxy; and (c) reducing the 7-oxo to 7P- hydroxy; thereby producing UDCA; wherein the foregoing steps may be performed simultaneously, sequentially, or in any order. In another embodiment, the method further comprises (a) contacting the 24-carboxylic acid on UDCA with a reagent that converts the acid to a derivative that can act as an acylating agent; and (b) reacting the derivative with taurine to form TUDCA.

[0122] While the methods of the current invention can generally be performed in any order, certain orders are preferred. Thus, for example, in any of embodiments four, five, seven, or eight, reduction step (a) preferably occurs before any other of the steps. Principal embodiments four and five and seven and eight can also define a series of “sequence embodiments” when A and B and Y are H. In one sequence embodiment when A and B and Y are H, step (a) is performed before step (b). In another sequence embodiment when A and B and Y are H, step (a) is performed before step (c). In another sequence embodiment when A and B and Y are H, step (a) is performed before steps (b) and (c). In another sequence embodiment when A and B and Y are H, step (b) is performed before step (c). In another sequence embodiment when A and B and Y are H, step (a) is performed before step (b) and step (b) is performed before step (c).

[0123] The steps in these sequence embodiments are preferably undertaken using the particular methodologies described further in this application. Thus, in any of the sequence embodiments, step (a) can be performed via catalytic hydrogenation, step (b) can be performed by contacting the 7-H with a 7P-hydroxylase, step (c) can be performed by contacting the 3-oxo with a 3a- ketoreductase, or all of steps (a), (b), and (c) can be performed as described in this sentence. In other sequence embodiments step (a) can be performed via catalytic hydrogenation, step (b) can be performed by contacting the 7-H with a 7P-hydroxylase, step (c) can be performed by contacting the 3-oxo with a 3a-ketoreductase, or all of steps (a), (b), and (c) can be performed as described in this sentence.

[0124] The catalytic hydrogenation is preferably carried out using flow chemistry and a trickle bed reactor, as described in the third principal embodiment. Thus, in one embodiment, the catalytic hydrogenation comprises: (a) pumping a solution comprising the compound of Formula la and an organic solvent through a trickle bed reactor packed with a hydrogenation catalyst; and (b) pumping hydrogen through the trickle bed reactor counter to or concurrently with the compound of Formula la.

[0125] The hydrogenation catalyst is preferably palladium defined by various parameters. In one embodiment, the hydrogenation catalyst is 3-30% palladium on activated carbon, preferably about 10% palladium on activated carbon. In another embodiment the hydrogenation catalyst is approximately 50% water wet palladium on activated carbon.

[0126] The solution preferably comprises from 5% to 20% (w:v) of the compound of Formula la relative to the organic solvent, more preferably about 10% (w:v). A particularly preferred organic solvent is dichloromethane or DCM. The solution also can contain a tertiary amine, preferably triethanolamine. In one embodiment, the hydrogenation is carried out in the presence of a tertiary amine, preferably TEA, at a molar ratio relative to the compound of Formula la of from 0.5:1 to 2: 1, preferably about 1 : 1.

[0127] Other conditions for the catalytic hydrogenation include hydrogen pressure, hydrogen molar ratio, residence time, and temperature. Thus, in one embodiment, the hydrogen gas is pumped into the reactor at a pressure of from 2 to 4 bar, preferably about 3 bar. In another embodiment the hydrogen gas and compound of formula la are pumped through the reactor at a molarratio (H2:compound Ia) offrom 1 : 1 to 2:1, preferably 1.1 : 1 to 1.5: 1. In another embodiment, the compound of Formula la has a residence time in the reactor of from 2 to 30 minutes at a temperature of from 30 to 80 °C, preferably 5 to 15 minutes at a temperature of 45 to 55 °C.

[0128] A particularly preferred substrate for the catalytic hydrogenation is KCEA.

[0129] Other preferred methods of catalytic hydrogenation are described, for example, in WO / 2023 / 081166 (published May 11, 2023), entitled Stereoselective Steroidal Reductions, by J. Reid et al. Preferred methods of 7P-hydroxylation are described, for example, in WO / 2022 / 115710 (published June 2, 2022), entitled Enzymatic Methods for Converting LCA and 3-KCA to UDCA and 3-KUDCA, by J. Reid et al., and WO / 2024 / 173367 (published August 22, 2024), entitled Recombinant Polypeptides for Biosynthesis of Ursodeoxycholic Acid (UDCA) by V. Zacharia et al. Preferred methods of 3a-ketoreduction are described, for example, in WO / 2018 / 036982 (published March 3, 2018), entitled Chemical-Biocatalytic Method for Producing Ursodeoxycholic Acid, by D. Bakonyi et al. The foregoing publications, and the referenced methods, are hereby incorporated by reference.

[0130] Other embodiments can also be defined by the identity of the product attained from each of the involved steps, and the purity of the product attained from each of the involved steps. Thus, when step (a) is performed via catalytic hydrogenation, step (a) preferably produces 3-KCA substantially free (> 90% or 95% purity w / w) of the corresponding 5a-isomer. When step (a) is performed by contacting the compound of Formula I or la with a reductase enzyme having 4,5- double bond reducing activity step (a) preferably produces 3-KCA free of the corresponding 5a- isomer. When step (b) is performed by contacting the 7-H with a 7P-hydroxylase, step (b) preferably produces 3-KUDCA free of the corresponding 7a-isomer. When step (c) is performed by contacting the 3 -oxo with a 3a-ketoreductase, step (c) preferably produces UDCA free of the corresponding 3P-isomer. The methods preferably achieve all of the foregoing purities.

[0131] Other embodiments can be defined by the identity of the product obtained from each of the involved steps and the yield (based on theoretical wt. %) achieved by each of the steps. Thus, further embodiments are defined when step (a) yields greater than 85%, 90%, 95%, 98%, 99%, or 99.5% 3-KCA. Other embodiments are defined when step (b) yields greater than 85%, 90%, 95%, 98%, 99%, or 99.5% 3-KUDCA. Other embodiments are defined when step (c) yields greater than 85%, 90%, 95%, 98%, 99%, or 99.5% UDCA. Thus, further embodiments are defined when step (a) yields greater than 85% 3-KCA, step (b) yields greater than 85% 3-KUDCA, and step (c) yields greater than 85% UDCA. Further embodiments are defined when step (a) yields greater than 90% 3-KCA, step (b) yields greater than 90% 3-KUDCA, and step (c) yields greater than 90% UDCA. Further embodiments are defined when step (a) yields greater than 95% 3-KCA, step (b) yields greater than 95% 3-KUDCA, and step (c) yields greater than 95% UDCA. Again, all of the foregoing yields are weight-based yields based on 100% theoretical conversion.

[0132] The methods can also be defined by the recovery of products from the various steps from the reaction medium before proceeding to the next step. Thus, further embodiments are defined when the product of step (a) is recovered from the reaction mixture. Other embodiments are defined when the product of step (b) is recovered from the reaction mixture. Other embodiments are defined when the product of step (c) is recovered from the reaction mixture. Other embodiments are defined when a combination of the products from steps (a), (b), and (c) are recovered from the reaction mixture. Other embodiments are defined when all of the products from steps (a), (b), and (c) are recovered from the reaction mixture.

[0133] A preferred method of converting the 21 -alcohol group to a c5 side chain terminating in 24-carboxylic acid, or converting the 21 -OX’ group to a 5-carbon cholanic acid side chain in which OX’ is replaced by CH2CO2H, is by (i) converting the 21-alcohol group or the 21-OX’ group to a leaving group; (ii) displacing the 21 -leaving group with dialkylmal onate under basic conditions; (iii) hydrolysis of both esters of the malonate group to give the dicarboxylic acid; and (iv) decarboxylation of the diacid to give the monoacid.

[0134] In other embodiments, when a reagent converts the acid to a derivative that can act as an acylating agent, the reagent is selected from the group consisting of SOCh, C1COCOC1, COCI2, SOBr2, TPP / NBS, I1112CO, EtOCOCl, (CHj^CCOCl, (Me0)2P(0)Cl, Diisopropylcarbodiimide, Diisopropylcarbodiimide + HOBt, Diisopropylcarbodiimide + HOSu, 2-Chloro-l- methylpyridinium iodide, and (Py)aP+Br (PFe)'. In another embodiment, the method further comprises contacting the 24-carboxylic acid on UDCA with means for converting the 24- carboxylic acid to a derivative that can act as an acylating agent, and reacting the derivative with taurine to form TUDCA, wherein the means corresponds to the reagents in the preceding sentence.

[0135] Methods for reducing the 7-oxo are disclosed, for example, in WO / 2023 / 081657 (Published 11 May 2023) (International Application No. PCT / US2022 / 079080) (entitled High Purity Non-Animal Derived UDCA). Reduction of the C22-C23 double bond can be accomplished, for example, via hydrogenation with Pd / carbon, as described in CN112375117A (Published 19 February 2021) (entitled Preparation method of lithocholic acid and intermediates thereof). The subject matter of WO / 2023 / 081657 and CN112375117A is incorporated herein by reference.

[0136] The methods of the current invention can be defined by various other parameters, including the purity of the derived material and the source of the substrate. Thus, in one embodiment the product of the reduction of the 4,5-double bond is isolated and purified to an isomeric purity relative to the corresponding 5a-isomer greater than 99.7%, 99.8%, 99.9%, 99.95%, 99.97%, or 99.98% (w / w). In another embodiment the product of the reduction of the 4,5-double bond, preferably the 4,5-double bond on KCEA or BA, occurs at an isomeric purity relative to the corresponding 5a-isomer of 100%.

[0137] In still further embodiments, applicable to any of the principal embodiments, the compound of Formula I, Formula la, Formula II, Formula Ila, Formula III or Formula IV comprises a 613C value corresponding to a plant derived molecule. Thus, in one embodiment the compound of Formula I, Formula la, Formula II, Formula Ila, Formula III or Formula IV comprises less than - 15%o, -17.5%o, -20%o, -22.5%o, or-25%o 8 I 3C relative to VPDB. Methods of analyzing 613C values are described, for example, in WO / 2023 / 081657 (published May 11, 2023), entitled High Purity Animal Derived UDCA, by J. Reid et al., the disclosure of which being hereby incorporated by reference.

[0138] Additional structural definitions

[0139] A substituent is “substitutable” if it comprises at least one carbon, sulfur, oxygen or nitrogen atom that is bonded to one or more hydrogen atoms. Thus, for example, hydrogen, halogen, and cyano do not fall within this definition. If a substituent is described as being “substituted,” a non-hydrogen substituent is in the place of a hydrogen substituent on a carbon, oxygen, sulfur or nitrogen of the substituent. Thus, for example, a substituted alkyl substituent is an alkyl substituent wherein at least one non-hydrogen substituent is in the place of a hydrogen substituent on the alkyl substituent.

[0140] If a substituent is described as being “optionally substituted,” the substituent may be either (1) not substituted, or (2) substituted. When a substituent is comprised of multiple moieties, unless otherwise indicated, it is the intention for the final moiety to serve as the point of attachment to the remainder of the molecule. For example, in a substituent A-B-C, moiety C is attached to the remainder of the molecule. If a plurality of substituents are described as being “independently selected” from a group, each substituent is selected independent of the other. Each substituent therefore may be identical to or different from the other substituent(s). In like manner, when a plurality of elements is defined using the same variable, and the variable is said to be “independently” selected from a plurality of sub-elements, each of the elements is defined by the variable without reference to how the variable is defined for the other elements. In any of the embodiments or subembodiments of this invention, a moiety which is optionally substituted may be alternatively defined as substituted with 0, 1, 2, or 3 substituents independently selected from halo, OH, amine, Ci-6 alkyl, Ci-6 alkoxy, Ci-6 hydroxyalkyl, CO(Ci-6 alkyl), SO3H, CHO, CO2H, CO2(Ci6alkyl), and C1-6 haloalkyl.

[0141] At various places in the present specification, substituents of compounds of the invention are disclosed in groups or in ranges. It is specifically intended that the invention include each and every individual sub-combination of the members of such groups and ranges. For example, the term “C1-6 alkyl” is specifically intended to individually disclose methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and Ce alkyl.

[0142] It is further appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.

[0143] As used herein, the term “alkyl” is meant to refer to a saturated hydrocarbon group which is straight-chained or branched. Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. In any of the embodiments or subembodiments of the present invention, an alkyl group can contain from 1 to about 20, from 2 to about 20, from 1 to about 10, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms.

[0144] As used herein, “aryl” refers to monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbons (including heteroaromatic hydrocarbons) such as, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, aryl groups have from 6 to about 20 carbon atoms.

[0145] As used herein, “halo” or “halogen” includes fluoro, chloro, bromo, and iodo.

[0146] As used herein, “alkoxy” refers to an -O-alkyl group. Example alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like.

[0147] As used herein “oxo” refers to =0.

[0148] The compounds described herein can be asymmetric (e.g., having one or more stereocenters). The description of a compound without specifying its stereochemistry is intended to capture mixtures of stereoisomers as well as each of the individual stereoisomer encompassed within the genus.

[0149] The present invention also includes salts of the compounds described herein, and when a compound is referred to herein, it will be deemed to include such salts unless specifically referred to as the free form. As used herein, “salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of suitable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The salts of the present invention include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

[0150] Finally, it will be understood that any of the novel compounds of the present invention (whether defined by a principal embodiment or a subembodiment or particular species) can be defined based on its purity and / or isolation from reaction media. Thus, in certain embodiments, the compounds are present in compositions at weight percentages greater than 10%, 50%, 90%, 95%, or 98% (w / w).

[0151] Methods

[0152] The invention may be carried out in the presence of a 4,5-reduction system, which is preferably a cellular system transformed to express At50StR or a variant thereof. The methods are useful in Escherichia species in general (e.g., E. coll), as well as bacterial cells that are harder to genetically manipulate but are otherwise valuable for production of chemicals, including Bacillus sp. (e.g., Bacillus subtilis), Streptomyces (e.g., Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces lividins, Streptomyces cinnamonensis, Streptomyces collinus, etc.), Cyanobacteria (e.g., Synechocystis spp., Prochlorococcus spp., Nostoc punctiforme, Calothrix spp., Aphanizomenon flos-aquae, Arthrospira platensis, etc.), and Corynebacteria (e.g., Corynebacteria glutamicum, Corynebacteria ammoniagenes, etc.), among others. The methods are further applicable in some embodiments to eukaryotic cells, such as yeasts including Pichia species in general (e.g., Pichia sp.) and other yeasts (e.g., Saccharomyces sp., Schizosaccharomyces sp., Kluyveromyces sp, etc.), filamentous fungi (e.g., Neurospora sp. and Aspergillus sp., Penicillium, etc.), Fusarium sp. (e.g. F. venenatum and F. graminearum, etc) , algae (e.g., Botryococcus, Chlorella, Dunaliella, Gracilaria, Pleurochrysis, and Sargassum, etc.), and plants. Preferred organisms are Escherichia coli or Pichia pastoris transformed to express At5pStR or a variant thereof.

[0153] In preferred embodiments, the methods of the current invention further comprise isolating the reduced substrate, or ester, amide, or salt thereof, from the system. By isolated it is meant that the reduced substrate is substantially free of the 4,5-reduction system and the reaction mixture in which the reduced substrate was produced. Thus, the reduced substrate is at least 90% pure, at least 95% pure, or at least 98% pure, when the weight of any residual reaction mixture is considered. Conversely, consistent with the usage of like terms herein, the reduced substrate will be free of no more than 10%, 5%, or 2% of residual reaction mixture. As used throughout this document, said purity percentages are all weight-based percentages.

[0154] In particularly preferred embodiments, the reduced substrate, or the ester, amide, or salt thereof, is produced substantially as a pure diastereomer. A “substantially pure diastereomer” refers to a diastereomer that is at least 98% or 99% pure when its corresponding diastereomer is considered. Conversely, a diastereomer that is “substantially free of its corresponding diastereomer” comprises no more than 1% or 2% of its corresponding diastereomer. As used throughout this document, purity percentages when expressed are weight-based percentages.

[0155] The process also can be defined based on the stereo- and regio- selectivity of the process. Thus, in any of the embodiments of the current invention, the At5pStR or At5pStR variant will stereo-selectively reduce the 4,5-double bond of the substrate to provide a product with 5P- stereochemistry at greater than 95%, 98%, 99%, 99.5%, or 99.8% efficiency, to the exclusion of 5 a- stereochemi stry .

[0156] In one embodiment, the invention provides UDCA, or the 24-ester, 24-amide, or salt thereof, produced by the methods of the current invention. In another embodiment, the invention provides UDCA, or the 24-ester, 24-amide, or salt thereof, produced by the methods of the current invention, comprising no more than 5%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, or 0.02% of any 5a- isomers.

[0157] In one embodiment the invention provides TUDCA, or a salt thereof, produced by the methods of the current invention. In another embodiment the invention provides TUDCA, or a salt thereof, produced by the methods of the current invention comprising no more than 5%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, or 0.02% of any 5a-isomers.

[0158] The methods can also be used to produce UDCA and TUDCA having the purities described in WO / 2023 / 081658 (Published 11 May 2023) (International Application No. PCT / US2022 / 079081) (entitled High Purity Non-Animal Derived TUDCA) and WO / 2023 / 081657 (Published 11 May 2023) (International Application No. PCT / US2022 / 079080) (entitled High Purity Non-Animal Derived UDCA), and according to the methods described in the foregoing patent publications. The disclosures of WO / 2023 / 081658 and WO / 2023 / 081657 are expressly incorporated herein by reference.

[0159] In one embodiment the methods of the current invention are carried out by contacting the compound of Formula I, Formula la, or Formula III, preferably KCEA or BA, with At5pStR or an analog thereof, and the method is carried out with whole cells that express the At50StR or analog thereof, or an extract or lysate of such cells.

[0160] In another embodiment the methods of the current invention are carried out by contacting the compound of Formula I, Formula la, or Formula III, preferably KCEA or BA, with At5pStR or an analog thereof, and the method is carried out with whole cells that express the At5pStR or analog thereof, or an extract or lysate of such cells, and the whole cells or extract or lysate of such whole cells are selected from recombinant Bacillus sp. (e.g., Bacillus subtilis), Streptomyces (e.g., Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces lividins, Streptomyces cinnamonensis, Streptomyces collimis, etc.), Cyanobacteria (e.g., Synechocystis spp., Prochlorococcus spp., Nostoc punctiforme, Calothrix spp., Aphanizomenon flos-aquae, Arthrospira platensis, etc.), and Corynebacteria (e.g., Corynebacteria glutamicum, Corynebacteria ammoniagenes, etc.), among others. The methods are further applicable in some embodiments to eukaryotic cells, such as yeasts including Pichia species in general (e.g., Pichia sp.) and other yeasts (e.g., Saccharomyces sp., Schizosaccharomyces sp., Kluyveromyces sp, etc.), filamentous fungi (e.g., Neurospora sp. and Aspergillus sp., Penicillium, etc.), Fusarium sp. (e g. F. venenatum and F. graminearum, etc), algae (e.g., Botryococcus, Chlorella, Dunaliella, Gracilaria, Pleurochrysis, and Sargassum , etc.), preferably Escherichia coll, Pichia pastoris or Saccharomyces cerevisiae.

[0161] In one embodiment the methods of the current invention are carried out by contacting the compound of Formula I, Formula la, or Formula III, preferably KCEA or BA, with At5pStR or an analog thereof, and the method is carried out with whole cells that express the At5pStR or analog thereof, or an extract or lysate of such cells, and the whole cells or extract or lysate of such whole cells are recombinant Escherichia coli cells.

[0162] In various additional embodiments the cells or extract or lysate of such cells are transformed by a At5pStR or analog thereof plasmid or are the progeny of cells transformed by a At5pStR or analog thereof plasmid, wherein the At5pStR or analog thereof is foreign to the cells.

[0163] Engineered At5fiStR or At5fiStR variant Enzymes

[0164] Enzymes having different properties than the enzyme sequences disclosed herein can be obtained by mutating the genetic material encoding the enzyme and identifying polynucleotides that express engineered enzymes with a desired property. These non-naturally occurring enzymes can be generated by various well-known techniques, such as in vitro mutagenesis or directed evolution. In some embodiments, directed evolution is an attractive method for generating engineered enzymes because of the relative ease of generating mutations throughout the whole of the gene coding for the polypeptide, as well as providing the ability to take previously mutated polynucleotides and subjecting them to additional cycles of mutagenesis and / or recombination to obtain further improvements in a selected enzyme property. Subjecting the whole gene to mutagenesis can reduce the bias that may result from restricting the changes to a limited region of the gene. It can also enhance generation of enzymes affected in different enzyme properties since distantly spaced parts of the enzyme may play a role in various aspects of enzyme function.

[0165] In mutagenesis and directed evolution, the parent or reference polynucleotide encoding the naturally occurring or wild type enzyme is subjected to mutagenic processes, for example random mutagenesis and recombination, to introduce mutations into the polynucleotide. The mutated polynucleotide is expressed and translated, thereby generating engineered enzymes with modifications to the polypeptide. As used herein, “modifications” include amino acid substitutions, deletions, and insertions, as well as chemical modifications of the amino acids. In like manner, a “variant” includes one or more “modifications.” An “analog” is a variant with retained or improved 5-fJ reduction properties. For example, an “analog” of At5pStR is a variant with retained or improved 5-P reduction activity when compared with the wild type enzyme of SEQ ID NO. 29. For example, analogs of At5pStR are polypeptides comprising or consisting of a sequence having greater than 80%, 85%, 90%, 95%, or 98% sequence identity with SEQ ID NO. 29, or a polypeptide comprising or consisting of a sequence containing up to 3, 5, 8, 10, 15, 25, 50, or 75 amino acid modifications with respect to SEQ ID NO: 29, wherein said polypeptides have retained or improved 5-P reduction activity when compared to SEQ ID NO: 29. Some analogs of At5pStR of defined sequence are defined below. Any one or a combination of modifications can be introduced into the naturally occurring enzymatically active polypeptide to generate engineered enzymes, which are then screened by various methods to identify polypeptides, and corresponding polynucleotides, having a desired improvement in a specific enzyme property.

[0166] Various embodiments of the current invention are defined based on the use of the wildtype At5pStR enzyme or a corresponding enzyme or fragment selected from SEQ ID NOs. 2, 6, 11, 16, 23, 30, 31, or 32, or an analog of one of the foregoing polypeptides. In any of these embodiments, the analog can comprise greater than 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any of the foregoing polypeptides. Alternatively or in addition, the analog can comprise up to 75, 50, 25, 15, 10, 8, 5, or 3 amino acid modifications to any of the foregoing enzymes

[0167] When modifications are made to any of the sequences described herein, in terms of homology or amino acid modifications, preferred modifications include modifications where Phenyl alanine (F) on positions 153 and / or 342 in SEQ ID NO: 29, or corresponding positions on related / corresponding amino acid sequences, is / are mutated to a sterically smaller hydrophobic amino acid, like Alanine (A), Glycine (G), Isoleucine (I) Leucine (L) or Valine (V), preferably Alanine (A).

[0168] The same logic applies to SEQ ID NO: 11, SEQ ID NO: 31 and SEQ ID NO: 32, where the Phenyl alanine (F) on positions corresponding to 153 and / or 342 in SEQ ID NO: 29 are already substituted with Alanine (A). In SEQ ID NO: 11, SEQ ID NO: 31 and SEQ ID NO: 32, the Alanine (A) in positions 157 and / or 346 in SEQ ID 11, 128 and / or 317 in SEQ ID 31, and 136 and / or 325 in SEQ ID 32 can be readily mutated to a sterically similar hydrophobic amino acid, like Glycine (G), Isoleucine (I) Leucine (L) or Valine (V), but preferably remains Alanine (A). In one embodiment, the compound of Formula I, la, or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of SEQ ID NO. 2, or a polypeptide comprising or consisting of a sequence having greater than 85%, 90%, 95%, or 98% sequence identity with SEQ ID NO. 2, or a polypeptide comprising or consisting of a sequence containing up to 3, 5, 8, 10, 15, 25, 50, or 75 amino acid modifications with respect to SEQ ID NO: 2.

[0169] In other embodiments the methods of the current invention are practiced by contacting the compound of Formula I, Formula la, or Formula III, preferably KCEA or BA, with a polypeptide comprising or consisting of SEQ ID NO. 6, or a polypeptide comprising or consisting of a sequence having greater than 80%, 85%, 90%, 95%, or 98% sequence identity with SEQ ID NO. 6, or a polypeptide comprising or consisting of a sequence containing up to 3, 5, 8, 10, 15, 25, 50, or 75 amino acid modifications with respect to SEQ ID NO: 6.

[0170] In other embodiments the methods of the current invention are practiced by contacting the compound of Formula I, Formula la, or Formula III, preferably KCEA or BA, with a polypeptide comprising or consisting of SEQ ID NO. 11, or a polypeptide comprising or consisting of a sequence having greater than 80%, 85%, 90%, 95%, or 98% sequence identity with SEQ ID NO. 11, or a polypeptide comprising or consisting of a sequence containing up to 3, 5, 8, 10, 15, 25, 50, or 75 amino acid modifications with respect to SEQ ID NO: 11.

[0171] In one embodiment, the compound of Formula I, la or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of SEQ ID NO. 16, or a polypeptide comprising or consisting of a sequence having greater than 80%, 85%, 90%, 95%, or 98% sequence identity with SEQ ID NO. 16, or a polypeptide comprising or consisting of a sequence containing up to 3, 5, 8, 10, 15, 25, 50, or 75 amino acid modifications with respect to SEQ ID NO: 16.

[0172] In one embodiment, the compound of Formula I, la or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of SEQ ID NO. 23, or a polypeptide comprising or consisting of a sequence having greater than 80%, 85%, 90%, 95%, or 98% sequence identity with SEQ ID NO. 23, or a polypeptide comprising or consisting of a sequence containing up to 3, 5, 8, 10, 15, 25, 50, or 75 amino acid modifications with respect to SEQ ID NO: 23.

[0173] In one embodiment, the compound of Formula I, la or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of SEQ ID NO. 29, or a polypeptide comprising or consisting of a sequence having greater than 80%, 85%, 90%, 95%, or 98% sequence identity with SEQ ID NO. 29, or a polypeptide comprising or consisting of a sequence containing up to 3, 5, 8, 10, 15, 25, 50, or 75 amino acid modifications with respect to SEQ ID NO: 29.

[0174] In other embodiments the methods of the current invention are practiced by contacting the compound of Formula I, Formula la, or Formula III, preferably KCEA or BA, with a polypeptide comprising or consisting of SEQ ID NO. 30, or a polypeptide comprising or consisting of a sequence having greater than 80%, 85%, 90%, 95%, or 98% sequence identity with SEQ ID NO. 30, or a polypeptide comprising or consisting of a sequence containing up to 3, 5, 8, 10, 15, 25, 50, or 75 amino acid modifications with respect to SEQ ID NO. 30.

[0175] In another embodiment, the compound of Formula I, la, or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of SEQ ID NO. 31, or a polypeptide comprising or consisting of a sequence having greater than 80%, 85%, 90%, 95%, or 98% sequence identity with SEQ ID NO. 31, or a polypeptide comprising or consisting of a sequence containing up to 3, 5, 8, 10, 15, 25, 50, or 75 amino acid modifications with respect to SEQ ID NO: 31.

[0176] In another embodiment the compound of Formula I, la, or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of SEQ ID NO. 32, or a polypeptide comprising or consisting of a sequence having greater than 80%, 85%, 90%, 95%, or 98% sequence identity with SEQ ID NO. 32, or a polypeptide comprising or consisting of a sequence containing up to 3, 5, 8, 10, 15, 25, 50, or 75 amino acid modifications with respect to SEQ ID NO: 32. 5-Reductase Environment

[0177] The enzymes of the present invention may be present within a cell, in the cellular medium, on an immobilized substrate, or in other forms, such as lysates and extracts of cells recombinantly designed to express the enzyme, or isolated preparations. The term “isolated polypeptide” refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it, e.g., protein, lipids, and polynucleotides. The term embraces polypeptides which have been removed or purified from their naturally-occurring environment or expression system (e g., host cell or in vitro synthesis). In some embodiments, the isolated enzymes are present in a substantially pure polypeptide composition. The term “substantially pure polypeptide” refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. Generally, a substantially pure enzyme composition will comprise about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. Conversely, the enzyme composition can be said to be free of no more than 40%, 30%, 20%, 10%, 5%, or 2% of other macromolecular species by mole or % weight. In some embodiments, the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a singular macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species.

[0178] Encoding Polynucleotide

[0179] An isolated polynucleotide encoding an enzyme may be manipulated in a variety of ways to provide for expression of the enzyme. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art. Guidance is provided in Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rdEd., Cold Spring Harbor Laboratory Press; and Current Protocols in Molecular Biology, Ausubel. F. ed., Greene Pub. Associates, 1998, updates to 2006.

[0180] Thus, in another aspect, the present disclosure is also directed to a recombinant expression vector comprising a polynucleotide encoding At5 StR or a At5pStR variant, and one or more expression regulating regions such as a promoter and a terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced. The various nucleic acid and control sequences may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the polypeptide at such sites. In creating the recombinant expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

[0181] The recombinant expression vector may be any vector (e.g., a plasmid or virus), which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the polynucleotide sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.

[0182] The expression vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a mini-chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell may be used. In particularly preferred embodiments, the plasmids or vectors of the current invention are under control of an A0X1 promoter and an A0X1 terminator sequence.

[0183] The term “control sequence” is defined herein to include all components, which are necessary or advantageous for the expression of a polypeptide of the present disclosure. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, transcriptional and translational stop signals, and a ribosome binding site (to stop translation). The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.

[0184] The term “operably linked” is defined herein is a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the expression of a polynucleotide and / or polypeptide. The control sequence may be an appropriate promoter sequence. The “promoter sequence” is a nucleic acid sequence that is recognized by a host cell for expression of the coding region. The promoter sequence contains transcriptional control sequences, which mediate the expression of the polypeptide. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0185] The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

[0186] Host Cells for Expression of At5 StR or At5fiStR variant

[0187] In another aspect, the present disclosure provides a host cell comprising the At5pStR or At5 StR variant of the present disclosure, the polynucleotides being operatively linked to one or more control sequences for expression of the enzymes in the host cell. Host cells for use in expressing the enzymes encoded by the expression vectors of the present invention are well known in the art and include Bacillus sp. (e.g., Bacillus subtilis), Streptomyces (e.g., Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces lividins, Streptomyces cinnamonensis, Streptomyces collinus, etc.), Cyanobacteria (e.g., Synechocystis spp., Prochlorococcus spp., Nostoc punctiforme, Calothrix spp., Aphanizomenon flos-aquae, Arthrospira platensis, etc.), and Corynebacteria (e.g., Corynebacteria glutamicum, Corynebacteria ammoniagenes, etc.), among others. The methods are further applicable in some embodiments to eukaryotic cells, such as yeasts including Pichia species in general (e.g., Pichia sp.) and other yeasts (e.g., Saccharomyces sp., Schizosaccharomyces sp., Kluyveromyces sp, etc.), filamentous fungi (e.g., Neurospora sp. and Aspergillus sp., Penicillium, etc.), Fusarium sp. (e.g. F. venenatum and F. graminearum, etc) , algae (e.g., Botryococcus, Chlorella, Dunaliella, Gracilaria, Pleurochrysis, and Sargassum, etc.), particularly E. Coli. Appropriate culture mediums and growth conditions in the foregoing cellular systems are well known in the art.

[0188] Polynucleotides for expression of the At5pStR or At5pStR variant may be introduced into cells by various methods known in the art. For the bacteria described herein, the typical process is by transformation (e.g. electroporation or calcium chloride mediated) or conjugation, or sometimes protoplast fusion. Various methods for introducing polynucleotides into cells will be apparent to the skilled artisan.

[0189] Reaction Conditions

[0190] In carrying out the stereoselective reduction described herein, the At5pStR or At5pStR variant may be added to the reaction mixture in the form of the purified enzymes (including immobilized variants), whole cells transformed with gene(s) encoding the enzymes, and / or cell extracts and / or lysates of such cells. The gene(s) encoding the engineered enzyme can be transformed into host cells separately or together into the same host cell.

[0191] Whole cells transformed with gene(s) encoding the enzymes, or cell extracts and / or lysates thereof, may be employed in a variety of different forms, including solid (e.g., lyophilized, spray- dried, immobilized, and the like) or semisolid (e.g., a crude paste). The cell extracts or cell lysates may be partially purified by precipitation (ammonium sulfate, polyethyleneimine, heat treatment or the like), followed by a desalting procedure prior to lyophilization (e.g., ultrafiltration, dialysis, and the like).

[0192] The quantities of reactants used in the reduction will generally vary depending on the quantities of enzyme substrate employed. The following guidelines can be used to determine the amounts of enzyme to use. Generally, steroid substrates are employed at a concentration of about 1 to 50 grams / liter using from about 50 mg / liter to about 5 g / liter of the reduction system. The weight ratio of the steroid to the reductase system in the reaction mixture is commonly from about 10: 1 to 200: 1. In the case of whole cells instead of isolated enzymes, the ratio of wet cells to substrate will commonly vary from about 1 : 10 to about 10: 1. Those having ordinary skill in the art will readily understand how to vary these quantities to tailor them to the desired level of productivity and scale of production.

[0193] The ene-reduction and other enzymatic conversions will commonly be performed in the presence of a cofactor, typically NADH and / or NADPH. The cofactor is usually present in a ratio of cofactor to substrate ranging from 1 :200 to 1 :20. The cofactor can also be recycled in a number of ways to improve the cost-efficiency of the process. In one embodiment, spent NADPH is regenerated by coupling with an NADPH regenerating enzyme. In another embodiment, spent NADH is regenerated by coupling with an NADH-regenerating enzyme. The order of addition of reactants is not critical. The reactants may be added together at the same time to a solvent (e.g., monophasic solvent, biphasic aqueous co-solvent system, and the like), or alternatively, some of the reactants may be added separately, and some together at different time points. For example, the reductase system may be added first to the solvent. Preferably, however, the enzyme preparation is added last.

[0194] Suitable conditions for carrying out the enzyme-catalyzed reactions described herein include a wide variety of conditions including contacting the enzymes and steroid substrate at an experimental pH and temperature and detecting product, for example, using the methods described in the Examples provided herein. Any of the embodiments can be carried out at a pH of from about pH 5 to about pH 10, preferably about 6 to about 9, more preferably from about 7.5 to 8.5, and most preferably about 8.

[0195] The reductase-catalyzed reactions described herein are generally carried out in a solvent. While water is most preferred, organic solvents such as acetone, isopropanol, ethyl acetate, butyl acetate, 1 -octanol, heptane, octane, methyl t-butyl ether (MTBE), toluene, and the like, and ionic liquids such as 1 -ethyl 4-methylimidazolium tetrafluoroborate, l-butyl-3-methylimidazolium tetrafluoroborate, l-butyl-3-methylimidazolium hexafluorophosphate, and the like, can be used in certain circumstances, either alone or in combination with water. In preferred embodiments, aqueous solvents, including water and aqueous co-solvent systems, are used. The solvent system is preferably greater than 50%, 75%, 90%, 95%, or 98% water, and in one embodiment is 100% water.

[0196] During the course of the reduction, the pH of the reaction mixture may change. The pH of the reaction mixture may be maintained at a desired pH or within a desired pH range by the addition of an acid or a base during the course of the reaction. Alternatively, the pH may be controlled by using a solvent that comprises a buffer. Suitable buffers to maintain desired pH ranges are known in the art and include, for example, phosphate buffer, triethanolamine buffer, and the like. Combinations of buffering and acid or base addition may also be used.

[0197] The reduction is typically carried out at a temperature in the range of from about 15°C to about 75°C. For some embodiments, the reaction is carried out at a temperature in the range of from about 20°C to about 55°C. In still other embodiments, it is carried out at a temperature in the range of from about 20°C to about 45°C. The reaction may also be carried out under ambient conditions. In various additional embodiments, the methods are carried out under conditions in which the weight ratio of the compound of Formula I or Formula III to the At5pStR or analog thereof in the reaction mixture is from about 5: 1 to 200: 1 or 10: 1 to 200: 1. In other embodiments, the methods are carried out in the presence of biomass, and the weight ratio of substrate to biomass (wet cell wt) is greater than 0.2, 1.0, or 5.0 g / g. In still further embodiments, the methods are carried out at a substrate concentration greater than 1.5 mM, 10 mM, 20 mM, 50 mM, or 100 mM.

[0198] The reaction is generally allowed to proceed until essentially complete, or near complete, reduction of substrate is obtained. Reduction of substrate to product can be monitored using known methods by detecting substrate and / or product. Suitable methods include gas chromatography, HPLC, and the like. Conversion yields of the steroid reduction product generated in the reaction mixture are generally greater than about 50%, may also be greater than about 60%, may also be greater than about 70%, may also be greater than about 80%, may also be greater than 90%, and can even be greater than about 97%.

[0199] The reductase product can be recovered from the reaction mixture and optionally further purified using methods that are known to those of skill in the art. Chromatographic techniques for isolation from the reductase system include, among others, silica gel chromatography, reverse phase chromatography high performance liquid chromatography, and ion exchange chromatography. Conditions for purifying a particular steroid will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those having skill in the art. A preferred method for product purification involves crystallization, generally after extraction into an organic solvent.

[0200] ADDITIONAL EMBODIMENTS FROM USSN 63 / 537,584

[0201] Embodiment 1) A method of reducing a 4,5-double bond on 3 -ketochol -4-enoic acid (KCEA) or a derivative thereof defined by Formula I, to preferentially give a 50-product, comprising contacting the compound of Formula I: with At50StR or an analog thereof having greater than 80%, 85%, 90%, 95%, or 98% sequence identity with At5pStR, in a reaction mixture under conditions suitable for reducing the 4,5-double bond, thereby producing the compound of Formula IE wherein: (a) A and B are OH and H respectively, H and OH respectively, H and H, or 7-oxo in combination; (b) X is C(O)OR1or C(O)NR1R2; and (c) R1and R2are independently hydrogen, a counterion when the compound is a carboxylate or amide salt, optionally substituted C1-20 alkyl, or optionally substituted aryl.

[0202] Embodiment 2) The method of embodiment 1, wherein the compound of Formula I is contacted with At50StR or an analog thereof having greater than 85%, 90%, 95%, or 98% sequence identity with At5pStR.

[0203] Embodiment 3) The method of embodiment 1 or 2, further comprising recovering the compound of Formula II from the reaction mixture.

[0204] Embodiment 4) The method of any of embodiments 1 -3, wherein the compound of Formula II is produced substantially or entirely as a pure diastereomer, such as compound lb, 3b, 4b, 5b, 6b, 7b, or 9b.

[0205] Embodiment 5) The method of any of embodiments 1-4, wherein A and B are 7-oxo in combination.

[0206] Embodiment 6) The method of any of embodiments 1-4, wherein A and B are 7-oxo in combination and X is C(O)OR1.

[0207] Embodiment 7) The method of any of embodiments 1-4, wherein A and B are H.

[0208] Embodiment 8) The method of any of embodiments 1-4, wherein A and B are H and X is C OR1.

[0209] Embodiment 9) The method of any of embodiments 1-4, wherein - is a single bond.

[0210] Embodiment 10) The method of any of embodiments 1-4, wherein - is a double bond.

[0211] Embodiment 11) The method of any of embodiments 1-4, wherein the compound of Formula I is selected from a compound of Formula la, 3a, 4a, 5a, 6a, 7a, or 9a, and the compound of Formula II is selected from a compound of Formula lb, 3b, 4b, 5b, 6b, 7b, or 9b, respectively, as defined elsewhere in this document.

[0212] Embodiment 12) The method of any of embodiments 1-4 and 11, wherein A and B are 7- oxo in combination, further comprising: (a) reducing the 3-oxo to 3a-hydroxy; (b) reducing the 7- oxo to 70-hydroxy, to produce UDCA or a precursor of UDCA; (c) when X is a C(0)0R' ester, hydrolyzing the ester to produce UDCA; and (d) when X is a C(O)NR'R2amide, hydrolyzing the amide to C(0)0H to produce UDCA; wherein the foregoing steps may be performed simultaneously, sequentially, or in any order.

[0213] Embodiment 13) The method of embodiment 12, wherein UDCA is formed, further comprising: (a) contacting the 24-carboxylic acid with a reagent that converts the acid to a derivative that can act as an acylating agent; and (b) reacting the derivative with taurine to form TUDCA.

[0214] Embodiment 14) The method of embodiment 13, wherein the reagent is selected from the group consisting of SOCh, C1C0C0C1, COCh, SOBr2, TPP / NBS, Im2C0, EtOCOCl, (CH3)3CCOC1, (MeO)2P(O)Cl, Diisopropylcarbodiimide, Diisopropylcarbodiimide + HOBt, Diisopropylcarbodiimide + HOSu, 2-Chloro-l -methylpyridinium iodide, and (Py) P+Br (PF6)-.

[0215] Embodiment 15) The method of embodiment 13, comprising contacting the 24-carboxylic acid with means for converting the 24-carboxylic acid to a derivative that can act as an acylating agent, and reacting the derivative with taurine to form TUDCA.

[0216] Embodiment 16) The method of any of embodiments 1 -4 and 1 1 , wherein A and B are H, further comprising: (a) reducing the 3-oxo to 3a-hydroxy; (b) hydroxylating the 7-H to 7 - hydroxy, to produce UDCA or a precursor of UDCA; (c) when X is a C(0)0R’ ester, hydrolyzing the ester to produce UDCA; and (d) when X is a C(0)NR'R2amide, hydrolyzing the amide to C(0)0H to produce UDCA; wherein the foregoing steps may be performed simultaneously, sequentially, or in any order.

[0217] Embodiment 17) The method of embodiment 16, further comprising: (a) contacting the 24- carboxylic acid with a reagent that converts the acid to a derivative that can act as an acylating agent; and (b) reacting the derivative with taurine to form TUDCA.

[0218] Embodiment 18) The method of embodiment 17, wherein the reagent is selected from the group consisting of SOCh, C1C0C0C1, COCh, SOBr2, TPP / NBS, Im2C0, EtOCOCl, (CH aCCOCl, (MeO)2P(O)Cl, Diisopropylcarbodiimide, Diisopropylcarbodiimide + HOBt, Diisopropylcarbodiimide + HOSu, 2-Chloro-l -methylpyridinium iodide, and (Py)3P+Br (PF6)-.

[0219] Embodiment 19) The method of embodiment 17, comprising contacting the 24-carboxylic acid with means for converting the 24-carboxylic acid to a derivative that can act as an acylating agent, and reacting the derivative with taurine to form TUDCA.

[0220] Embodiment 20) A method of reducing a 4,5-double bond on (20 S)-21 -hydroxy -20- methylpregn-4-en-3-one (BA) or a derivative thereof defined by Formula III, to preferentially give a 5p-product, comprising contacting the compound of Formula III: with At5pStR or an analog thereof having greater than 80%, 85%, 90%, 95%, or 98% sequence identity with At5pStR, in a reaction mixture under conditions suitable for reducing the 4,5-double bond, thereby producing the compound of Formula IV: wherein: (a) A and B are OH and H respectively, H and OH respectively, H and H, or 7-oxo in combination; and (b) X’ is hydrogen or a protecting group.

[0221] Embodiment 21) The method of any of embodiments 20-22, wherein the compound of Formula III is contacted with At5pStR or an analog thereof having greater than 85%, 90%, 95%, or 98% sequence identity with At5pStR.

[0222] Embodiment 22) The method of embodiment 20 or 21, further comprising recovering the compound of Formula IV from the reaction mixture.

[0223] Embodiment 23) The method of any of embodiments 20-22, wherein the compound of Formula IV is produced substantially as a pure diastereomer, such as compound 2b or 8b.

[0224] Embodiment 24) The method of any of embodiments 20-23, wherein X’ forms with the O to which it is attached an ester, an ether, a silyl ether, or an acetal. Embodiment 25) The method of any of embodiments 20-23, wherein A and B are H.

[0225] Embodiment 26) The method of any of embodiments 20-23, wherein A and B are H and X’ forms with the O to which it is attached an ester, an ether, a silyl ether, or an acetal.

[0226] Embodiment 27) The method of any of embodiments 20-23, wherein A and B are H and X’ is H.

[0227] Embodiment 28) The method of any of embodiments 20-23, wherein A and B are OH and H, respectively.

[0228] Embodiment 29) The method of any of embodiments 20-23, wherein A and B are OH and H, respectively, and X’ forms with the O to which it is attached an ester, an ether, a silyl ether, or an acetal.

[0229] Embodiment 30) The method of any of embodiments 20-23, wherein A and B are OH and H, respectively, and X’ is H.

[0230] Embodiment 31) The method of any of embodiments 20-23, wherein the compound of Formula III is selected from a compound of Formula 2a or 8a and the compound of Formula IV is selected from a compound of Formula 2b or 8b, respectively, as defined elsewhere in this document.

[0231] Embodiment 32) The method of any of embodiments 20-23 and 31, wherein A, B and X are H, further comprising: (a) converting the 21 -alcohol group to the 5-carbon cholanic acid side chain terminating in 24-carboxylic acid; (b) reducing the 3-oxo to 3a-hydroxy; (c) hydroxylating the 7-H to 7P-hydroxy, to produce UDCA; and (d) optionally contacting the 24-carboxylic acid of UDCA with a reagent that converts the carboxylic acid to a derivative that can act as an acylating agent and reacting the derivative with taurine to form TUDCA.

[0232] Embodiment 33) The method of any of embodiments 20-23 and 31, wherein A and B are OH and H, respectively, further comprising: (a) converting the 21 -alcohol group to the 5-carbon cholanic acid side chain terminating in 24-carboxylic acid; (b) reducing the 3-oxo to 3a-hydroxy, to produce UDCA; and (c) optionally contacting the 24-carboxylic acid of UDCA with a reagent that converts the carboxylic acid to a derivative that can act as an acylating agent and reacting the derivative with taurine to form TUDCA.

[0233] Embodiment 34) The method of embodiment 32 or 33, wherein the reagent is selected from the group consisting of SOC12, C1COCOC1, COCh, SOBr2, TPP / NBS, Im2C0, EtOCOCl, (CH 3CCOCI, (Me0)2P(0)Cl, Diisopropylcarbodiimide, Diisopropylcarbodiimide + HOBt, Diisopropylcarbodiimide + HOSu, 2-Chloro-l -methylpyridinium iodide, and (Py)3P+Br (PF6)-.

[0234] Embodiment 35) The method of embodiment 32 or 33, comprising contacting the UDCA with means for converting the 24-carboxylic acid to a derivative that can act as an acylating agent, and reacting the derivative with taurine to form TUDCA.

[0235] Embodiment 36) The method of any of the preceding embodiments, carried out with whole cells that express the At50StR or analog thereof, or an extract or lysate of such cells.

[0236] Embodiment 37) The method of any of the preceding embodiments, carried out with whole cells that express the At50StR or analog thereof, or an extract or lysate of such cells, wherein the whole cells or extract or lysate of such whole cells are selected from recombinant Escherichia coli, Pichia pastoris or Saccharomyces cerevisiae.

[0237] Embodiment 38) The method of any of the preceding embodiments, carried out with whole cells that express the At50StR or analog thereof, or an extract or lysate of such cells, wherein the whole cells or extract or lysate of such cells are recombinant Escherichia coli cells.

[0238] Embodiment 39) The method of embodiment 36, 37, or 38, wherein said cells or extract or lysate of such cells are transformed by a At50StR or analog thereof plasmid or are the progeny of cells transformed by a At50StR or analog thereof plasmid, wherein the At5pStR or analog thereof is foreign to the cells.

[0239] Embodiment 40) The method of any of the preceding embodiments, carried out at a temperature of from about 15 °C to about 75 °C.

[0240] Embodiment 41) The method of any of the preceding embodiments, carried out at a pH of from about pH 5 to about pH 10, preferably 6-9, 7.5-8.5, or 8.

[0241] Embodiment 42) The method of any of the preceding embodiments, wherein the weight ratio of the compound of Formula I or Formula III to the At5[3StR or analog thereof in the reaction mixture is from about 10:1 to 200:1.

[0242] Embodiment 43) The method of any of the preceding embodiments, wherein, when carried out in the presence of biomass, the weight ratio of substrate to biomass (wet cell wt) is > 0.2, 1, or 5 g / g-

[0243] Embodiment 44) The method of any of the preceding embodiments, carried out at a substrate concentration greater than 1.5 mM, 10 mM, 20 mM, 50 mM, or 100 mM. Embodiment 45) The method of any of embodiments 36-44, wherein the At5pStR comprises a modification of phenyl alanine (F) to an amino acid selected from the group consisting of Alanine (A), Glycine (G), Isoleucine (I) Leucine (L) and Valine (V), preferably Alanine (A), in the amino acid in position 153 and / or 342 in SEQ ID NO: 29, or the At5pStR analogue comprises a modification of phenyl alanine (F) to an amino acid selected from the group consisting of Alanine (A), Glycine (G), Isoleucine (I) Leucine (L) and Valine (V), preferably Alanine (A)in the amino acid corresponding to position 153 and / or 342 of SEQ ID NO: 29.

[0244] Embodiment 46) The method of any of embodiments 36-45, which is carried out in the presence of NADPH or NADH.

[0245] EXAMPLES

[0246] In the following examples, efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0247] EXAMPLE 1. PREPARATION OF KCEA FROM BISNORALCOHOL

[0248] Bromination of Bisnoralcohol:

[0249] To a stirred solution of bisnoralcohol (BA, 1 g, 3.02 mmol) in dichloromethane (DCM, 20 mL) was added PBn (0.34 mL, 3.63 mmol) at 0 °C. The mixture was warmed to room temperature and stirred for 3 hr, at which point TLC analysis showed complete conversion of starting material. The reaction mixture was quenched using ice water (10 mL), stirred for 15 min and the layers were separated. The aqueous layer was extracted in DCM (10 mL) and the combined organic phase was concentrated under reduced pressure to afford compound 1 as a yellow gummy oil (crude yield 1.2 g). 'H NMR (400 MHz, DMSO-d6): 6 5.61 (s, 1H), 3.56-3.51 (m, 1H), 3.45 (dd, J= 2.1 Hz and 1.1 Hz, 1H), 2.46-2.32 (m, 2H), 2.26-2.10 (m, 2H), 199-1.89 (m, 3H), 1.82-1.70 (m, 2H), 1.70- 0.82 (m, 18H), 0.70 (s, 3H) ppm.

[0250] Alkylation of diethyl malonate with compound 1 :

[0251] To a stirred solution of compound 1 (0.5 g, 1.27 mmol) in DMF (10 mL) was added diethyl malonate (0.58 mL, 3.812 mmol) at room temperature under N2 atmosphere. To this solution was added K2CO3 (526 mg, 3.812 mmol) followed by catalytic amounts of tetrabutylammonium hydrogen sulfate (TBAHS, 43 mg, 0.127 mmol). The reaction mixture was stirred at 75-80 °C for 48 hr and TLC analysis suggested complete conversion of starting material. After completion, the reaction mixture was quenched with ice water (10 mL) and the product was extracted using ethyl acetate (2 x 25 mL). The combined organic layer was washed with water (20 mL) and the organic phase was concentrated under reduced pressure to obtain compound 2 as gummy oil (crude yield 700 mg). 'H NMR (400 MHz, DMSO-d6): 8 5.61 (s, 1H), 4.0-4.20 (m, 4H), 3.50-3.42 (m, 1H), 2.43-2.30 (m, 2H), 2.27-2.10 (m, 2H), 2.11-1.90 (m, 3H), 1.89-1.70 (m, 2H), 1.62-0.80 (m, 27H), 0.63 (s, 3H) ppm. Mass analysis: m / z 473.40 [M+H]+was observed.

[0252] Hydrolysis of compound 2:

[0253] To a stirred solution of compound 2 (12 g, 25.38 mmol) in ethanol (120 mL) was added aq. potassium hydroxide solution (7.06 g in 120 mL water, 0.127 mol) at room temperature. The reaction mixture was heated to reflux for 2 hr and TLC analysis showed complete conversion of starting material. The ethanol was evaporated under reduced pressure and the solution was diluted with water (60 mL). The mixture was washed with DCM (60 mL, to remove impurities) and the pH of the aq. layer was adjusted to ~2 by using 6N HC1. The product was extracted using EtOAc (2 x 50 mL) and concentrated to dryness to afford compound 3 as a yellow solid (9.5 g).

[0254] Decarboxylation of compound 3 : To a 50 mL single neck round bottom flask was added compound 3 (1 g, 2.4 mmol.) in o- xylene (5 mL). The mixture was heated to reflux for 18 h and TLC analysis showed complete conversion of starting material. o-Xylene was removed under vacuum and the residue was treated with petroleum ether and the solid was filtered. The wet cake was washed with petroleum ether and dried under vacuum to afford KCEA as an off-white solid (0.5 g). 'H NMR (400 MHz, DMSO-D6): 8 11.95 (bs, 1H), 5.62 (s, 1H), 2.44-2.34 (m, 2H), 2.28-2.06 (m, 5H), 2.0-1.91 (m, 2H), 1.87-1.74 (m, 2H), 1.72-0.81 (m, 20H), 0.69 (s, 3H) ppm.

[0255] Example 2. 5ft-Reductions

[0256] The methods taught herein are employed to enzymatically reduce KCEA to 3-KCA and BA to compound 8 according to the following reaction sequences:

[0257] Molecular cloning of At5pStR and over-expression in E. coli.

[0258] At5pStR described by V. Herl et al. Biochimie 91 (2009) 517-525 is cloned into the pQE3x vector system for overexpression as His-tag fusion protein in E. coli. Over-expression of a recombinant form of At5pStR is initiated by the addition of 1 mM IPTG. Following Ni-NTA batch purification, the enzyme can be analyzed by SDS-PAGE. Under these conditions the recombinant form of At5pStR appears as a single band of around 45 000 Da.

[0259] Functional analysis of At5pStR and substrate selectivity.

[0260] The recombinant protein form of At50StR is incubated in the presence of NADPH and KCEA following the procedures described for the respective Digitalis enzymes in V. Herl et al. Phytochemistry 67 (2006) 225-231. In the presence of NADPH, KCEA is reduced to 3-KCA, having 5P-stereochemistry, to the exclusion of its corresponding 5a-isomer. The stereo-specificity of the enzyme reaction using KCEA as the substrate can be demonstrated by LC-MS.

[0261] In the presence of NADPH, BA is reduced to compound 8, having 5P-stereochemistry, to the exclusion of its corresponding 5a-isomer. The stereo-specificity of the enzyme reaction using BA as the substrate can be demonstrated by GC-MS.

[0262] EXAMPLE 3: PREPARATION 3-KCA FROM COMPOUND 8

[0263] Ketal protection of compound 8:

[0264] Compound 8 is dissolved in toluene in a flask fitted with a Dean-Stark trap. Ethylene glycol is added along with para-toluenesulfonic acid (pTSA) and the resulting solution is heated to reflux. Water is removed by use of the Dean-Stark trap. When the reaction is determined to be complete, an aqueous solution of sodium bicarbonate is added. After stirring for 10 minutes, the phases are separated and the toluene phase is concentrated under vacuum to provide compound 9 as a solid.

[0265] Bromination of compound 9:

[0266] To a stirred solution of compound 9 in dichloromethane (DCM, 20 mb) is added PBr? at 0 °C. The mixture is warmed to room temperature and stirred until the reaction is complete. The reaction mixture is quenched using a cold solution of sodium carbonate), stirred for 1 min and the layers are separated. The aqueous layer is extracted with DCM and the combined organic phase is concentrated under reduced pressure to afford compound 10 as an oil.

[0267] Alkylation of diethyl mal onate with compound 10:

[0268] To a stirred solution of compound 10 in DMF is added diethyl malonate at room temperature under N2 atmosphere. To this solution is added K2CO3 followed by a catalytic amount of tetrabutyl ammonium hydrogen sulfate. The reaction mixture is stirred at 75-80 °C until the reaction is complete. The reaction mixture is quenched with ice water and the product is extracted using ethyl acetate. The combined organic layer is washed with water and the organic phase is concentrated under reduced pressure to obtain compound 11 as an oil.

[0269] Hydrolysis of compound 11 :

[0270] To a stirred solution of compound 11 in ethanol is added aqueous potassium hydroxide solution at room temperature. The reaction mixture is heated to reflux until the reaction is complete. The ethanol is evaporated under reduced pressure and the solution is diluted with water. The mixture is washed with DCM to remove impurities and then fresh DCM is add. The pH of the aq. layer is adjusted to ~2 by using 12N HC1. The mixture is stirred until ketal hydrolysis is complete and then the phases are separated. The aqueous phase is extracted with DCM and the organic phases are combined. The solvent is removed under vacuum to provide compound 12 as a solid.

[0271] Decarboxylation of compound 12:

[0272] Compound 12 was dissolved in o-xylene and the mixture was heated to reflux. After all of the starting material was consumed, the o-Xylene was removed under vacuum and the residue was treated with petroleum ether. The solid was filtered and the wet cake was washed with petroleum ether and dried under vacuum to afford 3-KCA as solid.

[0273] EXAMP E 4: PREPARATION OF UDCA FROM 3-KCA

[0274]

[0275] 1. Pichia pastoris SAND121 is prepared as described in WO 2022 / 115710 A1 , example 16, and used according to Example 19 in the same document.

[0276] 2 3a-HSDH is 3a-Hydroxysteroid Dehydrogenase from Pseudomonas testosteroni (EC 1 1 1 50)

[0277] 3. GDH is D-Glucose Dehydrogenase from Bacillus megaterium (EC 1.1.1.47)

[0278] Hydroxylation of 3-KCA

[0279] Pichia pastoris SAND121 (from WO 2022 / 115710) is used to inoculate BMG medium, supplemented with 100 pg / mL nourseothricin and 100 pg / mL zeocin in an Erlenmeyer flask and incubated at 30°C, shaking at 250 RPM for 3 days. Aqueous 5-ALA solution (200 mM) and methanol containing 3-ketolithocholic acid (3-KCA) are added to the culture, then incubation is continued as before for 1 day. Methanol is added to the culture, then incubation is continued as before for 1 day. The mixture is acidified to pH 2-3 by addition of aq. HC1 and the resulting mixture is centrifuged. The solids are treated with acetone and centrifugation is repeated. The supernatant is concentrated under vacuum, which after removal of most of the acetone provides a slurry. The solids are removed by filtration and the product is washed with n-heptane and dried under vacuum to give compound 13 as a solid.

[0280] Reduction of the 3 -ketone of compound 13

[0281] To a 250 mL single neck round bottom flask were added compound 13 (1 g, 2.56 mmol), dextrose (1.3 g), P-NAD (33 mg) and 250 mM K2HPO4 buffer (70 mL) at room temperature. The mixture was stirred for 0.5 h to get a clear solution. 3a-HSDH (66 mg, from Pseudomonas testosteroni, EC 1.1.1.50) and GDH (2 mg, from Bacillus megaterium, EC 1.1.1.47) were added and the resulting mixture was stirred for 20 hr at room temperature. TLC analysis showed complete conversion of starting material. The reaction mixture was quenched with 2N HC1 solution until the pH reached 3-3.5, and then the product was extracted in EtOAc (3 x 50 mL) and concentrated under reduced pressure to obtain UDCA (900 mg, as a white solid). 'H NMR (500 MHz, MeOD): 5 3.44-3.53 (m, 2H), 2.30- 2.38 (m, 1H), 2.18-2.25 (m, 1H), 2.03 (dt, J = 6.5 Hz and 2.9 Hz, 1H), 1.95-1.79 (m, 5H), 1.56- 0.92 (m, 26H), 0.71 (s, 3H)

[0282] EXAMP E 5: SYNTHESIS OF TUDC A FROM UDCA _

[0283] UDCA (5 g, 12.736 mmol) was charged to a 100 mL single neck round bottom flask. Acetone (30 mL, 6 vol) was added, resulting in a solution. Triethylamine (TEA, 1.7 mL, 0.97 equiv.) was added and the solution was cooled to 0 °C. Ethyl chloroformate (1.34 g, 0.97 equiv.) was added and the resulting mixture was stirred for 4 h at room temperature under N2 atmosphere. The reaction mixture was filtered to remove triethylamine hydrochloride and the filtrate was added dropwise to an aqueous solution of taurine sodium salt (prepared by reacting 1.9 g taurine with 0.6 g NaOH in 3.7 mL water) at room temperature over a period of 20 minutes. The reaction was continued for another 1 h at room temperature, at which point TLC analysis showed complete conversion.

[0284] Cone. HC1 (1.5 mL), was added at room temperature to adjust the pH to ~1. After stirring for 1 h, the resulting solid was filtered (0.9 g). The filtrate was diluted with acetone, stirred for 36 h at room temperature and the resulting solid was filtered, washed with acetone (10 mL) and dried under vacuum to obtain TUDCA as a white solid (5.1 g, 80% yield).JH NMR (400 MHz, CD3OD): 8 3.62 (t, J = 6.8 Hz, 2H), 3.52-3.42 (m, 2H), 2.97 (t, J = 6.8 Hz, 2H), 2.38-2.28 (m, 1H), 2.20- 2.10 (m, 1H), 2.08-2.00 (m, 1H), 1.92-1.75 (m, 5H), 1.65-0.96 (m, 21H), 0.95 (s, 3H), 0.70 (s, 3H);13C NMR (100 MHz, CD3OD): 8 177.97, 72.12, 71.96, 47.45, 56.34, 50.78, 44.79, 44.46, 43.99, 41.52, 40.69, 38.56, 37.96, 37.47, 36.89, 36.07, 35.15, 33.34, 33.18, 30.99, 29.63, 27.91, 23.93, 22.37, 18.96, 12.64; ESIMS for C24H40O4 m / z 499.1 [M-H]+.

[0285] EXAMPLE 6: ADDITIONAL METHODS _

[0286] General methods

[0287] Isolation, handling and manipulation of DNA were carried out using standard methods (Green and Sambrook, 2012), which includes digestion with restriction enzymes, PCR, cloning techniques and transformation of bacterial cells. Green, M R., Sambrook J., Molecular Cloning: A Laboratory Manual (Fourth Edition). Cold Spring Harbor Laboratory, 2012.

[0288] Synthetic DNA was ordered from a commercial vendor, such as IDT (Coralville, Iowa, USA) or Twist Biosciences (South San Francisco, California, USA), as described in the examples. Gene sequences were supplied as linear DNA fragments, as described in the examples.

[0289] Sequencing of plasmid DNA was carried out by next-generation sequencing (NGS) by SNPsaurus (Eugene, Oregon, USA).

[0290] Transformation of P. pastoris

[0291] Competent cells were prepared by growing a single colony of Pichia pastoris NRRL Y- 11430 in 10 ml YPD overnight at 30 °C. 2-3 mL of the resulting culture was used to inoculate 50 ml of YPD in a 250-mL Erlenmeyer flask. This culture was grown at 30 °C to an ODeoo of 1.3-1.5. The cells were centrifuged at 2,000 RCF for 10 min at 4°C and resuspended in 10 ml of YPD, 2 mL IM HEPES (pH 8.0) and 25 pL IM DTT. The cell suspension was incubated at 30 °C for 15 minutes without shaking. Ice-cold deionized water was added to the cell suspension to a final volume of 50 mL. The cell suspension was centrifuged at 2,000 RCF for 10 min at 4°C. The pellet was suspended in 25 ml of ice-cold deionized water and centrifuged at 2,000 RCF for 10 min at 4°C. The pellet was suspended in 2 ml of ice-cold IM sorbitol and centrifuged at 2,000 RCF for 10 min at 4 °C. The pellet was resuspended in 0.3 ml of ice-cold IM sorbitol.

[0292] For transformation of Pichia, 40 pL of competent cells were mixed with 100 ng of linearized plasmid DNA and transformed by electroporation (1.5 kV, 200 Q, 25 pF) in an ice-cold electroporation cuvette. 1 ml of ice-cold IM sorbitol was added to the cuvette. The suspension was transferred to a sterile microcentrifuge tube and incubated at 30 °C for 2-3 hours with shaking at 250 PRM. The culture was spread on YPD agar containing 500 pg / mL zeocin.

[0293] Ene reductase activity assay

[0294] Enzymatic ene reductase activity was assayed as follows unless otherwise indicated. 1 mL reaction mixture in a 2-mL Eppendorf tube contained 25 pL sterile glucose solution (40 % w / v in deionized water), 25 pL NADPH solution (40 mM in reaction buffer), 2 pL GDH solution (3.75 U / pL), cell-free lysate in an appropriate volume of either reaction buffer or 50 mM potassium phosphate buffer (pH 8), as described in the examples and 50 pL substrate solution (20 mg / mL in DMSO). Reaction buffer or 50 mM potassium phosphate buffer (pH 8) was added to a total volume of 1 mL.

[0295] Reaction mixtures were mixed and incubated at 45 °C for 20 hours while shaking on an orbital platform at 600 RPM, after which they were extracted as described below and analyzed by HPLC.

[0296] HPLC analysis

[0297] Following solvent extraction of enzyme reactions or whole-cell bioconversion reactions as described in the Examples, the samples were analyzed for production of 3-KCA on an Agilent 1100 HPLC with a Gemini 3pm NX-C18 110A column (150 x 4.6 mm), fitted with a Gemini NX- 08 4 x 3.0 mm Security Guard Cartridge and operated at 45 °C. The mobile phase consisted of solvent A (0.1% phosphoric acid in water) and solvent B (0.1% phosphoric acid in acetonitrile) with a flow rate of 1.0 mL / minute. A gradient was run from 30% solvent B to 95% solvent B over 13 minutes. Samples were analyzed by UV at 196 nm.

[0298] Buffers and media

[0299] Buffers and media were made up as follows, unless otherwise indicated. To make 1 liter, all ingredients were dissolved in 800 mL deionized water in the order indicated. Deionized water is then added to the final volume, typically 1 liter unless the recipe called for post-sterilization additions, after which the liquid is sterilized by autoclaving at 121 °C for 20 minutes. Ingredients for volumes smaller than 1 liter were added in proportional amounts.

[0300] Breaking buffer contained 6 g / L sodium phosphate monobasic (pH 7.4), 1 mM EDTA and 50 mL / L glycerol. 1 mM PMSF was added prior to use. This buffer was not sterilized.

[0301] YPD contained 20 g / L peptone, 10 g / L yeast extract and 20 g / L glucose.

[0302] IM potassium phosphate buffer consisted of 132 mL of IM K2HPO4 and 868 mL IM KH2PO4 and is sterilized by autoclaving.

[0303] 20xP solution contained 268 g / L Na2HPO4, 136 g / L KH2PO4 and 66 g / L (NFL^SC

[0304] Reductase buffer contained 250 mM sucrose, 2 mM EDTA and 50 mM KH2PO4. pH was set to 8.0 with KOH prior to sterilization.

[0305] BMG contained 13.4 g / L yeast nitrogen base and 10 g / L glycerol in a total volume of 900 mL. 100 mL / L sterilized 1 M potassium phosphate buffer was added post-sterilization. BMMY contained 13.4 g / L yeast nitrogen base, 10 g / L yeast extract and 20 g / L peptone. 100 mL / L sterilized 1 M potassium phosphate buffer and 6.25 mL / L methanol were added poststerilization.

[0306] VPB (20P) contained 18 g / L vegetable peptone, 3 g / L yeast extract, 2.5 g / L K2HPO4 and 5 g / L NaCl. 20 mL / L sterile glucose (50 % w / v in deionized water) and 50 mL / L sterile 20xP solution were added post-sterilization.

[0307] 2x Pichia Bioconversion Media (2xPBM) contained 40 g / L yeast extract, 8 g / L tryptone and 50 mM potassium phosphate (pH 7.5) and was sterilized by filtration using a 0.22 pm syringe filter.

[0308] Solid media were made as described above, with the addition of 15 - 20 g / L agar prior to sterilization.

[0309] ITM 336 medium contained 4.25 g / L 85% H3PO4, 0.17 g / L CaSO4, 2.86 g / L K2SO4, 2.32 g / L MgSO4.7H2O, 0.64 g / L KOH, 50 g / L Glycerol, 4 drops AF204. 4.5 mL / L PTM1 solution was added post-sterilization. The pH of ITM 336 is increased to 6 with 30% NH4OH post-sterilization. Pichia Trace Metals Solution 1 (PTM1) contained 6 g / L Q1SO4.5H2O, 0.08 g / L Nal, 3 g / L MnSO4. H2O, 0.2 g / L Na2MoO. 2H2O, 0.02 g / L H3BO3, 0.5 g / L CoCi, 20 g / L ZnCl2, 65 g / L FeSO4.7H2O, 0.2 g / L biotin, 5 mL / L sulfuric acid. PTM1 is sterilized by filtration through a 0.22 pm filter.

[0310] Materials

[0311] DNA modification enzymes such as restriction endonucleases, PCR enzymes and others mentioned in the examples were purchased from New England Biolabs (NEB), Takara Bio or Promega. Media components, chemicals, and PCR primers were obtained from Sigma-Aldrich (Merck), Fischer Scientific (Thermo) or other commercial suppliers. 250-mL Thomson Ultrayield flasks and were obtained from BPES, UK.

[0312] Escherichia coli BL21 (DE3) was obtained from the BCCM strain collection (Ghent, Belgium), accession number LMBP 01455. This strain will be referred to as E. coli BL21 (DE3) in these examples.

[0313] Pichia pastoris was obtained from the ARS culture collection (Peoria, Illinois, USA), accession number NRRL Y-l 1430. This strain will be referred to as P. pastoris in these examples. EXAMPLE 6A: CONSTRUCTION OF AN ESCHERICHIA CO I STRAIN CAPABLE OF EXPRESSING A GENE

[0314] Plasmid p048-001, to express a gene with sequence SEQ ID NO. 1, encoding a protein with sequence SEQ ID NO. 2, under control of an inducible promoter and translationally fused to an N-terminal 6xHistidine tag, was constructed as follows. Wildtype DNA sequence of just the gene (without tags) is SEQ ID NO. 1. Wildtype amino acid sequence of just the enzyme (without tags) is SEQ ID NO. 2.

[0315] Plasmid pJK031, with sequence SEQ ID NO. 3, was linearised with restriction endonucleases Bcz / wHI-HF and AE I-HF (New England Biolabs) using standard methods, to be used as the linearised plasmid backbone.

[0316] Sequence SEQ ID NO. 4 was ordered as a linear synthetic DNA fragment (Twist Bioscience) and used in a Gibson assembly reaction (Gibson et al., 2009) with the linearised plasmid backbone. Gibson, D., Young, L., Chuang, RY. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6, 343-345 (2009). Correct assembly was verified by colony PCR using suitable primers and restriction digest, resulting in plasmid p048-001. Sequence integrity of p048-001 was verified by NGS.

[0317] E. coli BL21 (DE3) was transformed with plasmid p048-001 using standard methods, resulting in strain E. coli sp. SAND201.

[0318] EXAMPLE 6B : CONSTRUCTION OF AN A. COLI STRAIN CAPABLE OF EXPRESSING A CODON-OPTIMISED

[0319] GENE ENCODING ENE REDUCTASE FROM A. THALIANA. _

[0320] Plasmid p048-007, to express a gene with sequence SEQ ID NO. 5, encoding a protein with sequence SEQ ID NO. 6, under control of an inducible promoter, was constructed as follows. Plasmid pJK031, with sequence SEQ ID NO. 3 was linearised with restriction digestion enzymes Ndel and BamHI using standard methods, to be used as the linearised plasmid backbone.

[0321] Sequence SEQ ID NO. 7 was ordered as synthetic DNA (Integrated DNA Technologies) and amplified by PCR using primers SEQ ID NO. 8 and SEQ ID NO. 9, resulting in a 1184 bp fragment, followed by digestion with Ndel and BamHI using standard methods, to be used as the insert. The insert was ligated with the linearized plasmid backbone, resulting in plasmid p048-007. Correct assembly of the plasmid was verified by colony PCR using suitable primers and by NGS. E. coli BL21 (DE3) was transformed with plasmid p048-007 using standard methods, resulting in strain E. coli sp. SAND202.

[0322] EXAMP E 6C: CONSTRUCTION OF AN E. COLI STRAIN CAPABLE OF EXPRESSING A CODON-OPTIMISED GENE ENCODING A TRUNCATED ENE-REDUCTASE MUTANT Fl 53 A F342A FROM A. THALIANA. _

[0323] Plasmid p048-023, to express a gene with sequence SEQ ID NO. 10, encoding a protein with sequence SEQ ID NO. 11, under control of an inducible promoter, was constructed as follows. Plasmid pJK031, with sequence SEQ ID NO. 3 was cut with restriction digestion enzymes Ndei and / G / i / HI using standard methods, to be used as the linearised plasmid backbone.

[0324] Sequence SEQ ID NO. 12 was ordered as synthetic DNA (Integrated DNA Technologies) and amplified by PCR using primers SEQ ID NO. 13 and SEQ ID NO. 14, resulting in a 1167 bp fragment, followed by digestion with Ndei and BawHI using standard methods, to be used as the insert. The insert was ligated with the linearised plasmid backbone, resulting in plasmid p048-023. Correct assembly of the plasmid was verified by colony PCR on transformants using suitable primers and by NGS.

[0325] E. coli BL21 (DE3) was transformed with plasmid p048-023 using standard methods, resulting in strain A. coli sp. SAND203.

[0326] EXAMPLE 6D: CONSTRUCTION OF ANE. COLI STRAIN CAPABLE OF EXPRESSING A CODON-OPTI ISED GENE ENCODING ENE-REDUCTASE FROM A. THALIANA WITH N-TERMINAL UBIQUITIN SOLUBILITY TAG, _

[0327] Plasmid p048-003, to express a gene with sequence SEQ ID NO. 15 encoding a protein with sequence SEQ ID NO. 16, under control of an inducible promoter, was constructed as follows. Plasmid p048-001, as described in this document, was linearised by PCR using primers SEQ ID NO. 17 and SEQ ID NO. 18, resulting in a 6481 bp product, to be used as the linearised plasmid. Ubiquitin tag sequence SEQ ID NO. 19 was amplified using primer pair SEQ ID NO. 20 and SEQ ID NO. 21, resulting in a 325 bp product, to be used as the insert. The insert was cloned into the linearised plasmid using the using the Gibson assembly cloning method (Gibson et al. 2009), resulting in plasmid p048-003. Correct assembly of the plasmid was verified by colony PCR on transformants using suitable primers and by NGS. E. coli BL21 (DE3) was transformed with plasmid p048-003 using standard methods, resulting in strain E. coli sp. SAND204.

[0328] EXAMP E 6E : GENERATING CELL -FREE LYSATE OF E. COLI SPP. S AND201 , S AND202, S AND203 , SAND204, AND CHARACTERISATION OF ENE REDUCTASE ACTIVITY THEREIN. _

[0329] 50 mL VPB (20P) containing 50 pg / mL kanamycin in a 250-mL Erlenmeyer flask was inoculated with one of E. coli sp. SAND201, E. coli sp. SAND202, E. coli sp. SAND203, E. coli sp. SAND204, and incubated at 37 °C for 18 hours, shaking at 250 rpm (2.5 cm throw). Expression of the target gene and subsequent production of enzyme was induced by addition of IPTG to a final concentration of 0.1 mM. Each culture was incubated at 25 °C for 24 hours with shaking at 250 rpm (2.5 cm throw), to be used as the expression culture.

[0330] Broth from the expression culture was transferred to a 50 ml Falcon tube. Cells were harvested by centrifugation and suspended in 10 mL potassium phosphate buffer (100 mM, pH 8). Cells were lysed using sonication following the manufacturer’s instructions. Lysate was clarified by centrifugation and the supernatant transferred to a clean tube, to be used as the cell-free lysate. 50 pL cell-free lysate was used in an ene reductase activity assay (see General Methods). After 20 hours, 20 pL formic acid was mixed into each sample, after which it was extracted with 0.5 mL ethyl acetate. 100 pL of the solvent layer was evaporated under reduced pressure and the remaining pellet was first dissolved in 100 pL acetonitrile, then mixed with an equal volume of 50% acetonitrile in deionized water containing 0.1% phosphoric acid, to be used as the sample.

[0331] The sample was analyzed by HPLC-UV (see General Methods) and peaks with an identical retention time as seen with a 3-KCA standard run alongside were seen. A typical HPLC-UV trace for a sample drawn from an ene reductase activity assay using lysate of E. coli sp. SAND201 can be seen in figure 1. A typical HPLC-UV trace for a sample drawn from an ene reductase activity assay using lysate of E. coli sp. SAND203 can be seen in figure 2. A typical HPLC-UV trace for a sample drawn from an ene reductase activity assay using lysate of E. coli sp. SAND204 can be seen in figure 3. In all of these samples, no detectable levels of the 5a-isomer of 3-KCA were observed. EXAMPLE 6F: CONSTRUCTION OF A PICHIA PASTORIS STRAIN CAPABLE OF EXPRESSING A GENE ENCODING A REDUCTASE ROM ARABIDOPSIS TIIALIANA. _

[0332] Plasmid pSW498, to express a gene with sequence SEQ ID NO. 22, encoding a protein with sequence SEQ ID NO. 23, under control of an inducible promoter, was constructed as follows. Plasmid pSW496, with sequence SEQ ID NO. 24 was amplified by PCR using primers SEQ ID NO. 25 and SEQ ID NO. 26, resulting in a 4293 bp fragment, to be used as the linearised plasmid backbone.

[0333] A 1209 bp fragment was amplified from p048-001 by PCR using primers SEQ ID NO. 27 and SEQ ID NO. 28, to be used as the insert. The insert was coned into the linearised plasmid backbone using the Gibson assembly cloning method (Gibson et al., 2009), resulting in plasmid pSW498. Correct assembly of the plasmid was verified by colony PCR using suitable primers and by NGS.

[0334] Plasmid pSW498 was linearized with the restriction enzyme Pmel, to be used as linearized pSW498. Linearized pSW498 was used to transform P. pastoris (see General Methods). The resulting strain was labelled P. pastoris sp. SAND205.

[0335] EXAMPLE 6G: GENERATING CELL -FREE LYSATE OF P. PASTORIS SP. SAND205 AND CHARACTERISATION OF ENE REDUCTASE ACTIVITY THEREIN, _

[0336] One 50 mL Falcon tube containing 10 mL BMG and 500 pg / mL zeocin was inoculated with P. pastoris sp. SAND205. and incubated overnight at 28 °C, shaking at 250 RPM, to be used as the seed culture.

[0337] A 250 mL ultrayield shake flask containing 20 mL BMMY was inoculated with the seed culture to an ODeoo of 1 and incubated at 25 °C, shaking at 250 RPM for 24 hours, to be used as the expression culture. Twice daily, the expression culture was supplemented with additional methanol to a final concentration of 0.625%.

[0338] Cells from the expression culture were suspended in 100 pL breaking buffer to an ODeoo of 80. An equal volume of 0.5-mm diameter acid-washed glass beads was added and cells were lysed by 8 lysis cycles. Each lysis cycle consisted of vortex mixing for 30 seconds, followed by incubation on ice for 30 seconds. Cell debris was pelleted by centrifugation at 4 °C and the supernatant was transferred to a clean microcentrifuge tube, to be used as the cell-free lysate.

[0339] 50 pL cell-free lysate was used in an ene reductase activity assay (see General Methods). After 20 hours, 20 pL formic acid was mixed into each sample, after which it was extracted with 0.5 mL ethyl acetate. 100 pL of the solvent layer was evaporated under reduced pressure and the remaining pellet was first dissolved in 100 pL acetonitrile, then mixed with an equal volume of 50% acetonitrile in deionized water containing 0.1% phosphoric acid, to be used as the sample.

[0340] The sample was analysed by HPLC-UV (see General Methods) and peaks with an identical retention time as seen with a 3-KCA standard run alongside were seen. A typical HPLC-UV trace for a sample drawn from an ene reductase activity assay using lysate of P. pastoris sp. SAND205 can be seen in figure 4. The sample did not contain detectable levels of the 5a-isomer of 3-KCA.

[0341] EXAMP E 6H: WHOLE-CELL BIOCONVERSION OF KCEA TO 3-KCA USING P. PASTORIS SP.

[0342] SAND205

[0343] 400 mL BMG in a 2-L baffled Erlenmeyer flask was inoculated with P. pastoris sp. SAND205 to an OD OO of 0.02. This culture was incubated at 10 °C for 40 hours, then this culture was incubated at 30 °C for 24 hours with shaking at 250 RPM (2.5 cm radial throw), to be used as the seed culture.

[0344] The seed culture was used to inoculate 1 L ITM 336 in a 3-L stirred tank reactor to an ODeoo of 1.0, to be used as the batch phase culture. Incubation conditions were controlled by Applikon Biotechnology MyControl system with the following settings. The pH of the culture was held at 6.0 (deadband 0.1) with 30% NH4OH. Antifoam 204 was used to control foaming. The culture temperature was held at 30°C. An air flow 1 of vvm was sparged throughout this time using an L-shaped sparger. Dissolved oxygen was controlled at 30%.

[0345] A 40-mL sample withdrawn from the batch phase culture was centrifuged at 2000 RCF for 15 minutes. The pellet was suspended in 2xPBM to a final volume of 20 mL and transferred to an unbaffled 250-mL Erlenmeyer flask. To this suspension, 20 mL solution containing 50 mM potassium phosphate, pH 7.5, 1.53 g / L beta-cyclodextrin and 1 g / L KCEA was added, as well as 1% v / v methanol, to be used as the bioconversion culture. The bioconversion culture was incubated at 25 °C with shaking at 250 RPM (2.5 cm throw). After 4 hours of incubation, 1% (v / v) methanol was added, and incubation continued as before. After 20 hours of incubation, 0.8 mb of the bioconversion culture was mixed with 20 pL formic acid, and extracted with 0.8 mL ethyl acetate. After centrifugation, 200 pL of the solvent layer was evaporated under reduced pressure. The remaining pellet was first dissolved 100 pL acetonitrile, then mixed with an equal volume of 50% acetonitrile in deionized water containing 0.1% phosphoric acid, to be used as the sample.

[0346] The sample was analysed by HPLC-UV (see General Methods) and peaks with an identical retention time as seen with a 3-KCA standard run alongside were seen, as reported in figure 5. The sample did not contain detectable levels of the 5a-isomer of 3-KCA.

[0347] EXAMPLE 7: FLOW CHEMISTRY CONVERSION OF KCEA TO 3-KCA

[0348] KCEA 3 KCA TBA 3-KCA

[0349] Conditions: a 1) 10% Pd C in trickle bed reactor, TEA (1.0 eq), DCM ( 10 vol), 50 °C. 3.0 bar H2. 10 min residence in flow: 2) Distill to ~2.8 vol, add 3- picoline (6 vol) and MTBE (4 vol), then f-butylamine (1.1 eq) in MTBE (4 vol), 25 °C for 2 h, cool to 0 °C, filter; 3) IPA (2 vol), 3-picoline (0.1 vol), 70 °C, EtOAc (6 vol), cool to 25 °C, filter; b. 1) 1.5 M HC1, 25 °C. 2 h, filter; 2) MTBE (2 vol), 40 °C, w-heptane (6 vol), cool to 0 °C. filter

[0350] A solution of KCEA in DCM (10 vol) and TEA (1.0 eq) at 20-30 °C is pumped into a trickle bed reactor containing 20% Pd / C (50% water wet) with a slight molar excess of hydrogen gas (3.0 bar) with a 10-minute residence time at 50 °C. The combined 3-KCA containing effluent is distilled to 2.6-2.9 vol and diluted with 3-picoline (6 vol) and MTBE (4 vol). A solution of tertbutylamine (TBA, 1.1 eq) in MTBE (4 vol) is added at 20-30 °C. After 2 h, the mixture is cooled to -5 to 5 °C and held for NLT 1 h. The solids are filtered, washed with MTBE (1 vol) and dried under vacuum at 40-45 °C to afford the TBA salt of 3-KCA (3-KCA TBA). The sample is combined with IPA (2 vol) and 3-picoline (0.1 vol), heated to 65-75 °C and held for 0.5 h. EtOAc (6 vol) is added dropwise over ~0.5 h and the mixture is held at 65-75 °C for -1 h. The mixture is cooled to 20-30 °C, held for 0.5 h and filtered. The filter cake is washed with EtOAc (1 vol) and dried under vacuum at 40-45 °C to afford 3-KCA TBA.

[0351] 3-KCA TBA is combined with 1.5 M HC1 (6 vol), held at 20-30 °C for 2 h and filtered. The filter cake is washed with water (2 vol) and dried under vacuum at 40-50 °C to afford 3-KCA. The sample is combined with MTBE (2 vol), heated to 40-50 °C and n-heptane (6 vol) is added dropwise over ~0.5 h. After 0.5 h, the mixture is cooled to -5 to 5 °C, held for -1 h, filtered, washed with n-heptane (2 vol) and dried under vacuum at 40-45 °C to afford 3-KCA.

[0352] At5pStR Sequence Listings

[0353] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

CLAIMS1) A method of reducing a 4,5-double bond on 3-ketochol-4-enoic acid (KCEA) or a derivative thereof defined by Formula I, to preferentially give a 5P-product, comprising contacting the compound of Formula I:with At5pStR or an analog thereof having greater than 70%, 80%, 85%, 90%, 95%, or 98% sequence identity with At5pStR, in a reaction mixture under conditions suitable for reducing the 4,5-double bond, thereby producing the compound of Formula II:wherein: a) A and B are OH and H respectively, H and OH respectively, H and H, or 7-oxo in combination; b) X is C(O)OR1or C(O)NR1R2; and c) R1and R2are independently hydrogen, a counterion when the compound is a carboxylate or amide salt, optionally substituted C1-20 alkyl, or optionally substituted aryl.2) A method of reducing a 4,5-double bond on 3-ketochol-4-enoic acid (KCEA) or a derivative thereof defined by Formula la, to preferentially give a 5p-product, comprising contacting the compound of Formula la:with At5pStR or an analog thereof having greater than 70%, 80%, 85%, 90%, 95%, or 98% sequence identity with At5pStR, in a reaction mixture under conditions suitable for reducing the 4,5-double bond, thereby producing the compound of Formula Ila:wherein, in Formulas la and Ila: a) A and B are OH and H respectively, H and OH respectively, H and H, or 7-oxo in combination; b) X is C(O)OR1or C(O)NR1R2; c) Y is II or C(O)OR'; d) R1and R2are independently hydrogen, a counterion when the compound is a carboxylate or amide salt, optionally substituted C1-20 alkyl, or optionally substituted aryl; and e) the C22-C23 bond is a single or double bond.3) A method of reducing a 4,5-double bond on 3-ketochol-4-enoic acid (KCEA) or a derivative thereof defined by Formula la, to preferentially give a 5P-product, comprising pumping a solution comprising the compound of Formula la:and an organic solvent through a trickle bed reactor packed with a hydrogenation catalyst, and pumping hydrogen through the trickle bed reactor counter to or concurrently with the solution, thereby producing the compound of Formula Ila:wherein, in Formulas la and Ila: a) A and B are OH and H respectively, H and OH respectively, H and H, or 7-oxo in combination; b) X is C(O)OR1or C(O)NR1R2; c) Y is II or C(O)OR'; d) R1and R2are independently hydrogen, a counterion when the compound is a carboxylate or amide salt, optionally substituted C1-20 alkyl, or optionally substituted aryl; and e) the C22-C23 bond is a single or double bond.4) A method of making UDCA or TUDCA comprising: a) reducing the 4,5-double bond of a compound of Formula la:to produce the compound of Formula Ila:Ila wherein, in Formulas la and Ila, A and B are OH and H respectively, H and H, or 7-oxo in combination; X is C(O)OR1or C(O)NR1R2; Y is H or C(O)OR1; R1and R2are independently hydrogen, a counterion when the compound is a carboxylate or amide salt, optionally substituted C1-20 alkyl, or optionally substituted aryl; and the C22-C23 bond is a single or double bond; b) when A and B are H, hydroxylating the 7-H to 7p~hydroxy; c) reducing the 3 -oxo to 3a-hydroxy; d) when A and B are 7-oxo in combination, reducing the 7-oxo to 7P-hydroxy; e) when the C22-23 bond is a double bond, reducing it to a single bond; f) when Y is C(O)OR' and R1is H, decarboxylating C23 to convert Y to H; g) when Y is C(O)OR1and R1is optionally substituted C1-20 alkyl or optionally substituted aryl, hydrolyzing Y to convert R1to H and decarboxylating C23 to convert Y to H; h) when X is a C(O)OR1ester, hydrolyzing the ester to C(O)OH; and i) when X is a C(O)NR'R2amide and NR'R2is not taurine, hydrolyzing the amide to C(O)OH; thereby producing UDCA or TUDCA, wherein the foregoing steps may be performed simultaneously, sequentially, or in any order.) A method of making TUDCA comprising: a) reducing the 4,5-double bond of a compound of Formula la:to produce the compound of Formula Ila:Ila wherein, in Formula la and Ila, A and B are OH and H respectively, H and H, or 7-oxo in combination; X is C(O)OR1or C(O)NR1R2; Y is H or C(O)OR1; R1and R2are independently hydrogen, a counterion when the compound is a carboxylate or amide salt, optionally substituted C1-20 alkyl, or optionally substituted aryl; and the C22-C23 bond is a single or double bond; b) when A and B are H, hydroxylating the 7-H to 7P-hydroxy; c) reducing the 3 -oxo to 3a-hydroxy; d) when A and B are 7-oxo in combination, reducing the 7-oxo to 7p~hydroxy; e) when the C22-23 bond is a double bond, reducing it to a single bond; f) when Y is C(O)OR' and R1is H, decarboxylating C23 to convert Y to H; g) when Y is C(O)OR' and R1is optionally substituted C1-20 alkyl or optionally substituted aryl, hydrolyzing Y to convert R1to H and decarboxylating C23 to convert Y to H; h) when X is a C(O)OR’ ester, hydrolyzing the ester to C(O)OH; and i) when X is a C(O)NR1R2amide, hydrolyzing the amide to C(O)OH; thereby producing UDCA, further comprising:j) contacting 24-carboxylic acid of UDCA with a reagent that converts the 24- carboxylic acid to a derivative that can act as an acylating agent; and k) reacting the derivative with taurine; thereby forming TUDCA, wherein steps (a) through (i) may be performed simultaneously, sequentially, or in any order.6) A method of reducing a 4,5-double bond on (20S)-21-hydroxy-20-methylpregn-4-en-3-one(BA) or a derivative thereof defined by Formula III, to preferentially give a 50-product, comprising contacting the compound of Formula III:with At5pStR or an analog thereof having greater than 70%, 80%, 85%, 90%, 95%, or 98% sequence identity with At5pStR, in a reaction mixture under conditions suitable for reducing the 4,5-double bond, thereby producing the compound of Formula IV:wherein, in Formulas III and IV: a) A and B are OH and H respectively, H and OH respectively, H and H, or 7-oxo in combination; and b) X’ is hydrogen or a protecting group.7) A method of making UDCA comprising: a) reducing the 4,5-double bond of a compound of Formula III:to produce the compound of Formula IV:IV wherein, in Formulas III and IV, A and B are OH and H respectively, H and H, or 7-oxo in combination, and X’ is hydrogen or a protecting group; further comprising: b) when A and B are H, hydroxylating the 7-H to 7 -hydroxy; c) reducing the 3 -oxo to 3a-hydroxy; d) converting the 21 -OX’ group to a 5-carbon cholanic acid side chain in which OX’ is replaced by CH2CO2H; and e) when A and B are 7-oxo in combination, reducing the 7-oxo to 7P-hydroxy; thereby producing UDCA, wherein the foregoing steps may be performed simultaneously, sequentially, or in any order.8) A method of making TUDCA comprising: a) reducing the 4,5-double bond of a compound of Formula III:to produce the compound of Formula IV:wherein, in Formulas III and IV, A and B are OH and H respectively, H and H, or 7-oxo in combination, and X’ is hydrogen or a protecting group; further comprising: b) when A and B are H, hydroxylating the 7-H to 7p~hydroxy; c) reducing the 3 -oxo to 3a-hydroxy; d) converting the 21-OX’ to a 5-carbon cholanic acid side chain in which OX’ is replaced by CH2CO2H carboxylic acid; e) when A and B are 7-oxo in combination, reducing the 7-oxo to 7p~hydroxy; f) contacting the 24-carboxylic acid with a reagent that converts the 24-carboxylic acid to a derivative that can act as an acylating agent; and g) reacting the derivative with taurine; thereby forming TUDCA, wherein steps (a) through (g) may be performed simultaneously, sequentially, or in any order, except that step (d) must be done before step (f), and step (g) must be performed after step (f).9) The method of claim 5 or 8, wherein the reagent is selected from the group consisting of SOC12, C1COCOC1, COC12. SOBr2, TPP / NBS, Im2CO, EtOCOCl, (CH3)3CCOC1, (Me0)2P(0)Cl, Diisopropylcarbodiimide, Diisopropylcarbodiimide + HOBt, Diisopropylcarbodiimide + HOSu, 2-Chloro-l -methylpyridinium iodide, and (Py)3P+Br ( -10) The method of claim 5 or 8, wherein the reagent comprises means for converting the 24- carboxylic acid to a derivative that can act as an acylating agent.11) The method of any of claims 1-5, wherein A and B are 7-oxo in combination.12) The method of any of claims 1-5, wherein A and B are 7-oxo in combination, X is C(O)OR', Y is H, and the C22-C23 bond is a single bond.13) The method of any of claims 1-5, wherein A and B are 7-oxo in combination, X is C(O)OH, Y is H, and the C22-C23 bond is a single bond.14) The method of any of claims 1 -5, wherein A and B are H.15) The method of any of claims 1 -5, wherein A and B are H, X is C(O)OR1, Y is H, and the C22-C23 bond is a single bond.16) The method of any of claims 1-5, wherein A and B are H, X is C(O)OH, Y is H, and theC22-C23 bond is a single bond.17) The method of any of claims 1-5, wherein A and B are OH and H, respectively.18) The method of any of claims 1-5, wherein A and B are OH and H, respectively, X is C(O)OR', Y is H, and the C22-C23 bond is a single bond.19) The method of any of claims 1-5, wherein A and B are OH and H, respectively, X is C(O)OH, Y is H, and the C22-C23 bond is a single bond.20) The method of any of claims 1-5, wherein the C22-C23 bond is a single bond.21) The method of any of claims 1-5, wherein the C22-C23 bond is a double bond.22) The method of any of claims 1-5, wherein the compound of Formula I or la is selected from a compound of Formula la, 3a, 4a, 5a, 6a, 7a, or 9a, and the compound of Formula II or Ila is selected from a compound of Formula lb, 3b, 4b, 5b, 6b, 7b, or 9b, respectively:23) The method of any of claims 1-5, wherein A and B are 7-oxo in combination and Y is H, further comprising: a) reducing the 3 -oxo to 3a-hydroxy; b) reducing the 7-oxo to 7 -hydroxy; c) when the C22-C23 bond is a double bond, reducing it to a single bond; d) when X is a C(O)OR' ester, hydrolyzing the ester; and e) when X is a C(O)NR' R2amide, hydrolyzing the amide; thereby producing UDCA, wherein the foregoing steps may be performed simultaneously, sequentially, or in any order.24) The method of any of claims 1-5, wherein A, B, and Y are H, further comprising: a) reducing the 3 -oxo to 3a-hydroxy; b) when the C22-C23 bond is a double bond, reducing it to a single bond; c) hydroxylating the 7-H to 7 -hydroxy; d) when X is a C(O)OR' ester, hydrolyzing the ester; and f) when X is a C(O)NR'R2amide, hydrolyzing the amide to C(O)OH; thereby producing UDCA, wherein the foregoing steps may be performed simultaneously, sequentially, or in any order.25) The method of any of claims 1-5, wherein A and B are OH and H, respectively, and Y is H, further comprising: a) reducing the 3 -oxo to 3a-hydroxy;b) when the C22-C23 bond is a double bond, reducing it to a single bond; c) when X is a C(O)OR1ester, hydrolyzing the ester; and d) when X is a C(O)NR1R2amide, hydrolyzing the amide; thereby producing UDCA, wherein the foregoing steps may be performed simultaneously, sequentially, or in any order.26) The method of claim 4 or 5 in which A and B and Y are H, wherein: a) step (a) is performed before step (b); b) step (a) is performed before step (c); c) step (a) is performed before steps (b) and (c); d) step (b) is performed before step (c); or e) step (a) is performed before step (b) and step (b) is performed before step (c).27) The method of claim 4 or 5 in which A and B and Y are H, wherein step (a) is performed before step (b) and step (b) is performed before step (c).28) The method of claim 26 or 27, wherein: a) step (a) is performed by contacting the compound of Formula I or la with a reductase enzyme having 4,5-double bond reducing activity; b) step (b) is performed by contacting the 7-H with a 7P-hydroxylase; or c) step (c) is performed by contacting the 3 -oxo with a 3a-ketoreductase; or d) a combination of steps (a) - (c).29) The method of claim 26 or 27, wherein: a) step (a) is performed via catalytic hydrogenation; b) step (b) is performed by contacting the 7-H with a 7P-hydroxylase; or c) step (c) is performed by contacting the 3 -oxo with a 3a-ketoreductase; or d) a combination of steps (a) - (c).30) The method of claim 29, wherein step (a) comprises: a) pumping a solution comprising the compound of Formula I or la and an organic solvent through a trickle bed reactor packed with a hydrogenation catalyst; and b) pumping hydrogen through the trickle bed reactor counter to or concurrently with the compound of Formula I or la.31) The method of claim 3 or 30 wherein the hydrogenation catalyst is palladium.32) The method of claim 3 or 30, wherein the hydrogenation catalyst is 3-30% palladium onactivated carbon, preferably about 10% palladium on activated carbon.33) The method of claim 3 or 30, wherein the hydrogenation catalyst is approximately 50% water wet palladium on activated carbon.34) The method of claim 3 or 30, wherein the solution comprises from 5% to 20% (w:v) of the compound of Formula I or la relative to the organic solvent, preferably about 10% (w:v).35) The method of claim 3 or 30, wherein the organic solvent comprises DCM.36) The method of claim 3 or 30, wherein the hydrogenation is carried out in the presence of a tertiary amine, preferably TEA.37) The method of claim 3 or 30, wherein the hydrogenation is carried out in the presence of a tertiary amine, preferably TEA, at a molar ratio relative to the compound of Formula I or la of from 0.5: 1 to 2: 1, preferably about 1 : 1.38) The method of claim 3 or 30, wherein the hydrogen gas is pumped into the reactor at a pressure of from 2 to 4 bar, preferably about 3 bar.39) The method of claim 3 or 30, wherein the hydrogen gas and compound of formula la are pumped through the reactor at a molar ratio (H2: compound la) of from 1 :1 to 2: 1, preferably 1.1 : 1 to 1.5: 1.40) The method of claim 3 or 30, wherein the compound of Formula la has a residence time in the reactor of from 2 to 30 minutes at a temperature of from 30 to 80 °C, preferably 5 to 15 minutes at a temperature of 45 to 55 °C.41) The method of claim 3 or 30, wherein the compound of Formula la is KCEA.42) The method of claim 28, wherein: a) step (a) produces 3-KCA free of the corresponding 5a-isomer; b) step (b) produces 3-KUDCA free of the corresponding 7a-isomer; and c) step (c) produces UDCA free of the corresponding 3p-isomer.43) The method of claim 29, wherein: a) step (a) produces 3-KCA substantially free (> 90% or 95%) of the corresponding 5a-isomer; b) step (b) produces 3-KUDCA free of the corresponding 7a-isomer; and c) step (c) produces UDCA free of the corresponding 3p-isomer.44) The method of claim 42 or 43, wherein: a) step (a) yields greater than 85%, 90%, 95%, 98%, 99%, or 99.5% 3-KCA;b) step (b) yields greater than 85%, 90%, 95%, 98%, 99%, or 99.5% 3-KUDCA; and / or c) step (c) yields greater than 85%, 90%, 95%, 98%, 99%, or 99.5% UDCA.45) The method of claim 44, further comprising recovering from the reaction mixture: a) the product of step (a); b) the product of step (b); c) the product of step (c); or d) a combination thereof.46) The method of any of claims 6-8, wherein X’ forms with the O to which it is attached an ester, an ether, a silyl ether, or an acetal.47) The method of any of claims 6-8, wherein A and B are H.48) The method of any of claims 6-8, wherein A and B are H and X’ forms with the O to which it is attached an ester, an ether, a silyl ether, or an acetal.49) The method of any of claims 6-8, wherein A and B are H and X’ is H.50) The method of any of claims 6-8, wherein A and B are 7-oxo in combination.51) The method of any of claims 6-8, wherein A and B are 7-oxo in combination and X’ forms with the O to which it is attached an ester, an ether, a silyl ether, or an acetal.52) The method of any of claims 6-8, wherein A and B are 7-oxo in combination and X’ is H.53) The method of any of claims 6-8, wherein A and B are OH and H, respectively.54) The method of any of claims 6-8, wherein A and B are OH and H, respectively, and X’ forms with the O to which it is attached an ester, an ether, a silyl ether, or an acetal.55) The method of any of claims 6-8, wherein A and B are OH and H, respectively, and X’ is H.56) The method of any of claims 6-8, wherein the compound of Formula III is selected from a compound of Formula 2a or 8a and the compound of Formula IV is selected from a compound of Formula 2b or 8b, respectively:57) The method of any of claims 6-8, wherein A and B form 7-oxo in combination, further comprising: a) converting the 21-OX’ group to a 5-carbon cholanic acid side chain in which OX’ is replaced by CH2CO2H; b) reducing the 3 -oxo to 3a-hydroxy; and c) reducing the 7-oxo to 7p-hydroxy; thereby producing UDCA, wherein the foregoing steps may be performed simultaneously, sequentially, or in any order.58) The method of any of claims 6-8, wherein A and B are H, further comprising: a) converting the 21-OX’ group to a 5-carbon cholanic acid side chain in which OX’ is replaced by CH2CO2H; b) reducing the 3 -oxo to 3a-hydroxy; and c) hydroxylating the 7-H to 70-hydroxy; thereby producing UDCA, wherein the foregoing steps may be performed simultaneously, sequentially, or in any order.59) The method of any of claims 6-8, wherein A and B are OH and H, respectively, further comprising: a) converting the 21-OX’ group to a 5-carbon cholanic acid side chain in which OX’ is replaced by CH2CO2H; and b) reducing the 3 -oxo to 3a-hydroxy; thereby producing UDCA, wherein the foregoing steps may be performed simultaneously, sequentially, or in any order.60) The method of claim 7 or 8 in which A and B are H, wherein: a) step (a) is performed before step (b); b) step (a) is performed before step (c); c) step (a) is performed before steps (b) and (c); d) step (b) is performed before step (c); ore) step (a) is performed before step (b) and step (b) is performed before step (c).61) The method of claim 7 or 8 in which A and B are H, wherein step (a) is performed before step (b) and step (b) is performed before step (c).62) The method of claim 60 or 61, wherein: a) step (a) is performed via catalytic hydrogenation or by contacting the compound of Formula I or la with a reductase enzyme having 4,5-double bond reducing activity; b) step (b) is performed by contacting the 7-H with a 7P-hydroxylase; or c) step (c) is performed by contacting the 3 -oxo with a 3 a -ketoreductase; or d) a combination thereof.63) The method of claim 60 or 61, wherein: a) step (a) is performed via catalytic hydrogenation. b) step (b) is performed by contacting the 7-H with a 7P-hydroxylase; and c) step (c) is performed by contacting the 3-oxo with a 3a-ketoreductase.64) The method of claim 60 or 61, wherein: a) step (a) is performed by contacting the compound of Formula I or la with a reductase enzyme having 4,5-double bond reducing activity; b) step (b) is performed by contacting the 7-H with a 7P-hydroxylase; and c) step (c) is performed by contacting the 3-oxo with a 3a-ketoreductase.65) The method of claim 63, wherein: a) the product of step (a) is substantially free (>90% or >95%) of the corresponding 5a-isomer; b) the product of step (b) is free of the corresponding 7a-isomer; and c) the product of step (c) is free of the corresponding 3P-isomer.66) The method of claim 64, wherein: a) the product of step (a) is free of the corresponding 5a-isomer; b) the product of step (b) is free of the corresponding 7a-isomer; and c) the product of step (c) is free of the corresponding 3p-isomer.67) The method of claim 65 or 66, wherein: a) the yield from step (a) is greater than 85%, 90%, 95%, 98%, 99%, or 99.5%; b) the yield from step (b) is greater than 85%, 90%, 95%, 98%, 99%, or 99.5%; or c) the yield from step (c) is greater than 85, 90%, 95%, 98%, 99%, or 99.5%.68) The method of claim 67, further comprising recovering from the reaction mixture: a) the product of step (a); b) the product of step (b); c) the product of step (c); or d) a combination thereof.69) The method of any of the preceding claims, wherein the product of the reduction of the4,5-double bond is isolated and purified to an isomeric purity relative to the corresponding 5a-isomer greater than 98%, 99%, 99.5%, 99.7%, 99.8%, 99.9%, 99.95%, 99.97%, or 99.98%.70) The method of any of the preceding claims, wherein the product of the reduction of the4,5-double bond is isolated and purified to an isomeric purity relative to the corresponding 5a-isomer greater than 99.7%, 99.8%, 99.9%, 99.95%, 99.97%, or 99.98%.71) The method of any of the preceding claims, wherein the product of the reduction of the4,5-double bond, preferably 3-KCA or Compound 2b, occurs at an isomeric purity relative to the corresponding 5a-isomer of 100%.72) The method of any of the preceding claims, wherein the compound of Formula I, la, or III comprises a 6, 3C value corresponding to a plant derived molecule, preferably comprising less than -15%o, -17.5%o, -20%o, -22.5%o, or -25%o 513C relative to VPDB.73) The method of any of claims 1-72, wherein the compound of Formula I, la, or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of SEQ ID NO. 2, or a polypeptide comprising or consisting of a sequence having greater than 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO. 2.74) The method of any of claims 1-72, wherein the compound of Formula I, la, or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of a sequence containing up to 3, 5, 8, 10, 15, 25, 50, or 75 amino acid modifications with respect to SEQ ID NO: 2.75) The method of claim 73 or 74, wherein the polypeptide comprises a modification selected from F167A, F356A, and a combination thereof.76) The method of any of claims 1-72, wherein the compound of Formula I, la, or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of SEQID NO. 32, or a polypeptide comprising or consisting of a sequence having greater than 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO. 6.77) The method of any of claims 1-72, wherein the compound of Formula I, la, or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of a sequence containing up to 3, 5, 8, 10, 15, 25, 50, or 75 amino acid modifications with respect to SEQ ID NO: 6.78) The method of claim 76 or 77, wherein the polypeptide comprises a modification selected from Fl 73 A, F362A, and a combination thereof.79) The method of any of claims 1-72, wherein the compound of Formula I, la, or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of SEQ ID NO. 11, or a polypeptide comprising or consisting of a sequence having greater than 80%, 85%, 90%, 95%, or 98% sequence identity with SEQ ID NO. 11.80) The method of any of claims 1-72, wherein the compound of Formula I, la, or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of a sequence containing up to 3, 5, 8, 10, 15, 25, 50, or 75 amino acid modifications with respect to SEQ ID NO: 11.81) The method of claim 79 or 80, wherein the polypeptide comprises Al 57, A346, or a combination thereof.82) The method of any of claims 1-72, wherein the compound of Formula I, la, or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of SEQ ID NO. 16, or a polypeptide comprising or consisting of a sequence having greater than 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO. 16.83) The method of any of claims 1-72, wherein the compound of Formula I, la, or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of a sequence containing up to 3, 5, 8, 10, 15, 25, 50, or 75 amino acid modifications with respect to SEQ ID NO: 16.84) The method of claim 82 or 83, wherein the polypeptide comprises a modification selected from F242A, F431 A, and a combination thereof.85) The method of any of claims 1-72, wherein the compound of Formula I, la, or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of SEQID NO. 23, or a polypeptide comprising or consisting of a sequence having greater than 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO. 23.86) The method of any of claims 1-72, wherein the compound of Formula I, la, or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of a sequence containing up to 3, 5, 8, 10, 15, 25, 50, or 75 amino acid modifications with respect to SEQ ID NO: 23.87) The method of claim 85 or 86, wherein the polypeptide comprises a modification selected from Fl 53 A, F342A, and a combination thereof.88) The method of any of claims 1-72, wherein the compound of Formula I, la or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of SEQ ID NO. 29, or a polypeptide comprising or consisting of a sequence having greater than 80%, 85%, 90%, 95%, or 98% sequence identity with SEQ ID NO. 29.89) The method of any of claims 1-72, wherein the compound of Formula I, la, or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of a sequence containing up to 3, 5, 8, 10, 15, 25, 50, or 75 amino acid modifications with respect to SEQ ID NO: 29.90) The method of claim 88 or 89, wherein the polypeptide comprises a modification selected from Fl 53 A, F342A, and a combination thereof.91) The method of any of claims 1-72, wherein the compound of Formula I, la, or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of SEQ ID NO. 30, or a polypeptide comprising or consisting of a sequence having greater than 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO. 30.92) The method of any of claims 1-72, wherein the compound of Formula I, la, or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of a sequence containing up to 3, 5, 8, 10, 15, 25, 50, or 75 amino acid modifications with respect to SEQ ID NO: 30.93) The method of claim 91 or 92, wherein the polypeptide comprises a modification selected from F128A, F317A, and a combination thereof.94) The method of any of claims 1-72, wherein the compound of Formula I, la, or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of SEQID NO. 31 , or a polypeptide comprising or consisting of a sequence having greater than 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO. 31.95) The method of any of claims 1-72, wherein the compound of Formula I, la, or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of a sequence containing up to 3, 5, 8, 10, 15, 25, 50, or 75 amino acid modifications with respect to SEQ ID NO: 31.96) The method of claim 94 or 95, wherein the polypeptide comprises A128, A317, or a combination thereof.97) The method of any of claims 1-72, wherein the compound of Formula I, la, or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of SEQ ID NO. 32, or a polypeptide comprising or consisting of a sequence having greater than 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO. 32.98) The method of any of claims 1-72, wherein the compound of Formula I, la, or III, preferably KCEA or BA, is contacted with a polypeptide comprising or consisting of a sequence containing up to 3, 5, 8, 10, 15, 25, 50, or 75 amino acid modifications with respect to SEQ ID NO: 32.99) The method of claim 97 or 98, wherein the polypeptide comprises A136, A325, or a combination thereof.100) The method of claim 1 or 2, wherein KCEA is contacted with a polypeptide comprising or consisting of SEQ ID NO. 32, or a polypeptide comprising or consisting of a sequence containing up to 3, 5, 8, 10, 15, 25, 50, or 75 amino acid modifications with respect to SEQ ID NO: 32, preferably containing a modification selected from Al 36, A325, and a combination thereof.101) The method of claim 4 or 5, wherein KCEA is contacted with a polypeptide comprising SEQ ID NO. 32, or a polypeptide comprising or consisting of a sequence containing up to 3, 5, 8, 10, 15, 25, 50, or 75 amino acid modifications with respect to SEQ ID NO: 32, preferably containing a modification selected from A136, A325, and a combination thereof.102) The method of claim 6, wherein BA is contacted with a polypeptide comprising SEQ ID NO. 32, or a polypeptide comprising or consisting of a sequence containing up to 3, 5, 8,10, 15, 25, 50, or 75 amino acid modifications with respect to SEQ ID NO: 32, preferably containing a modification selected from A136, A325, and a combination thereof.103) The method of claim 7 or 8, wherein BA is contacted with a polypeptide comprising SEQ ID NO. 32, or a polypeptide comprising or consisting of a sequence containing up to 3, 5, 8, 10, 15, 25, 50, or 75 amino acid modifications with respect to SEQ ID NO: 32, preferably containing a modification selected from A136, A325, and a combination thereof.104) The method of the preceding claims wherein the compound is also contacted with NADPH or NADH.105) The method of any of the preceding claims wherein, when the compound of Formula I, la, or III, preferably KCEA or BA, is contacted with At50StR or an analog thereof, the method is carried out with whole cells that express the At5 StR or analog thereof, or an extract or lysate of such cells.106) The method of any of the preceding claims wherein, when the compound of Formula I, la, or III, preferably KCEA or BA, is contacted with At5 StR or an analog thereof, the method is carried out with whole cells that express the At50StR or analog thereof, or an extract or lysate of such cells, and the whole cells or extract or lysate of such whole cells are selected from recombinant Escherichia coli, Pi chia past oris or Saccharomyces cerevisiae.107) The method of claim 105 or 106, wherein said cells or extract or lysate of such cells are transformed by a plasmid encoding At5 StR or analog thereof or are the progeny of such cells, wherein the At5pStR or analog thereof is foreign to the cells.108) The method of any of the claims 105-107, carried out at a temperature of from about 15 °C to about 75 °C.109) The method of any of claims 105-108, carried out at a pH of from about pH 5 to about pH 10, preferably 6-9, 7.5-8.5, or 8.110) The method of any of claims 105-109, wherein the weight ratio of the compound of Formula I, la, or III to the At5pStR or analog thereof in the reaction mixture is from about 5: 1 to 200: 1 or 10:1 to 200: 1.111) The method of any of claims 105-110, wherein, when carried out in the presence of biomass, the weight ratio of substrate to biomass (wet cell wt) is > 0.2, 1, or 5 g / g.112) The method of any of claims 105-111, carried out at a substrate concentration greater than 1.5 mM, 10 mM, 20 mM, 50 mM, or 100 mM.113) A compound of Formula II obtained by the method of claim 1 .114) A compound of Formula Ila obtained by the method of claim 2 or 3.115) Compound lb, 3b, 4b, 5b, 6b, 7b, or 9b obtained by the method of claim 1, 2, or 3.116) 3-KCA obtained by the method of claim 1, 2, or 3.117) UDCA obtained by the method of claim 4 or 7.118) TUDCA obtained by the method of claim 5, 6 or 8.119) A compound of Formula IV obtained by the method of claim 6.120) Compound 2b or 8b obtained by the method of claim 6.121) A compound of Formula Ila:comprising a 813C value corresponding to a plant derived molecule, preferably comprising less than -15%o, -17.5%o, -20%o, -22.5%o, or -25%o 813C relative to VPDB, and an isomeric purity relative to the corresponding 5a-isomer greater than 98%, 99%, 99.5%, 99.7%, 99.8%, 99.9%, 99.95%, 99.97%, or 99.98%; wherein: a) A and B are OH and H respectively, H and H, or 7-oxo in combination; b) X is C(O)OR1or C(O)NR1R2; c) Y is H or C^OR1; d) R1and R2are independently hydrogen, a counterion when the compound is a carboxylate or amide salt, optionally substituted C1-20 alkyl, or optionally substituted aryl; and e) the C22-C23 bond is a single or double bond.122) The compound of claim 121, having an isomeric purity relative to the corresponding 5a- isomer of 100%.123) The compound of claim 121 or 122, wherein A and B are 7-oxo in combination.124) The compound of claim 121 or 122, wherein A and B are 7-oxo in combination, X is C(O)OR’, Y is H, and the C22-C23 bond is a single bond.125) The compound of claim 121 or 122, wherein A and B are H.126) The compound of claim 121 or 122, wherein A and B are H, X is C(O)OR1, Y is H, and the C22-C23 bond is a single bond.127) The compound of claim 121 or 122, which is 3-KCA.128) The compound of claim 121 or 122, wherein A and B are OH and H, respectively.129) The compound of claim 121 or 122, wherein A and B are OH and H, respectively, X is C(O)OR', Y is H, and the C22-C23 bond is a single bond.130) The compound of claim 121 or 122, wherein the compound of Formula Ila is selected from a compound of Formula lb, 3b, 4b, 5b, 6b, 7b, or 9b:31) The compound of any of claims 121-130, obtained by a method comprising contacting the compound of Formula la:with At5pStR or an analog thereof having greater than 70%, 80%, 85%, 90%, 95%, or 98% sequence identity with At50StR, in a reaction mixture under conditions suitable for reducing the 4,5-double bond, thereby producing the compound of Formula Ila:wherein, in Formulas la and Ila: a) A and B are OH and H respectively, H and OH respectively, H and H, or 7-oxo in combination; b) X is C(O)OR1or C(O)NR1R2; c) Y is H or C(O)OR'; d) R1and R2are independently hydrogen, a counterion when the compound is a carboxylate or amide salt, optionally substituted C1-20 alkyl, or optionally substituted aryl; ande) the C22-C23 bond is a single or double bond.132) A compound of Formula IV:comprising a 813C value corresponding to a plant derived molecule, preferably comprising less than -15%o, -17.5%o, -20%o, -22.5%o, or -25%o 813C relative to VPDB, and an isomeric purity relative to the corresponding 5a-isomer greater than 98%, 99%, 99.5%, 99.7%, 99.8%, 99.9%, 99.95%, 99.97%, or 99.98%; wherein: a) A and B are OH and H respectively, H and H, or 7-oxo in combination; and b) X’ is hydrogen or a protecting group.133) The compound of claim 132, having an isomeric purity relative to the corresponding 5a- isomer of 100%.134) The compound of claim 132 or 133, wherein X’ forms with the O to which it is attached an ester, an ether, a silyl ether, or an acetal.135) The compound of claim 132 or 133, wherein A and B are H.136) The compound of claim 132 or 133, wherein A and B are H and X’ forms with the O to which it is attached an ester, an ether, a silyl ether, or an acetal.137) The compound of claim 132 or 133, wherein A and B are H and X’ is H.138) The compound of claim 132 or 133, wherein A and B are 7-oxo in combination.139) The compound of claim 132 or 133, wherein A and B are 7-oxo in combination and X’ forms with the O to which it is attached an ester, an ether, a silyl ether, or an acetal.140) The compound of claim 132 or 133, wherein A and B are 7-oxo in combination and X’ is H.141) The compound of claim 132 or 133, wherein A and B are OH and H, respectively.142) The compound of claim 132 or 133, wherein A and B are OH and H, respectively, and X’ forms with the O to which it is attached an ester, an ether, a silyl ether, or an acetal.143) The compound of claim 132 or 133, wherein A and B are OH and H, respectively, and X’ is H.144) The compound of claim 132 or 133, wherein the compound of Formula IV is selected from a compound of Formula 2b or 8b:145) The compound of any of claims 132-144, obtained by a method comprising contacting the compound of Formula III:with At5pStR or an analog thereof having greater than 70%, 80%, 85%, 90%, 95%, or 98% sequence identity with At5pStR, in a reaction mixture under conditions suitable for reducing the 4,5-double bond, thereby producing the compound of Formula IV:wherein, in Formulas III and IV: a) A and B are OH and H respectively, H and OH respectively, H and H, or 7-oxo in combination; and b) X’ is hydrogen or a protecting group.

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