Recombinant polypeptides for biosynthesis of ursodeoxycholic acid (UDCA)

Abstract

The present disclosure relates to recombinant polypeptides with cytochrome P450 (CYP) activity capable of selectively catalyzing the conversion of precursor compounds, LCA or 3-KCA, to the product compounds, UDCA or 3-KUDCA, and the engineering of these polypeptides for heterologous expression in yeast allowing for enhanced fermentative bioproduction of these product compounds.

Description

RECOMBINANT POLYPEPTIDES FOR BIOSYNTHESIS OF URSODEOXYCHOLIC ACID (UDCA) CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority of U.S. Provisional Patent Application Number 63 / 586,338, filed September 28, 2023, and U.S. Provisional Patent Application Number 63 / 484,926, filed February 14, 2023, the entirety of each of which is hereby incorporated by reference herein. FIELD

[0002] The present disclosure relates to recombinant polypeptides with cytochrome P450 (CYP) activity capable of selectively catalyzing the conversion of precursor compounds, LCA or 3-KCA, to the product compounds, UDCA or 3-KUDCA, and the engineering of these polypeptides for heterologous expression in yeast allowing for improved fermentative bioproduction of these product compounds.   REFERENCE TO SEQUENCE LISTING

[0003] The official copy of the Sequence Listing is submitted concurrently with the specification via USPTO Patent Center as an WIPO Standard ST.26 formatted XML file with file name “13421-020WO1.xml”, a creation date of February 13, 2024, and a size of 1,355,138 bytes. This Sequence Listing filed via USPTO Patent Center is part of the specification and is incorporated in its entirety by reference herein.  BACKGROUND

[0004] Ursodeoxycholic acid (UDCA) is a secondary bile acid that has high therapeutic value in the treatment of cholestatic liver diseases. UDCA also has cytoprotective and anti-inflammatory properties and is being pursued as a potential therapeutic to treat neurological, cardiovascular, and inflammatory bowel diseases. Currently, UDCA is primarily harvested from animal bile (black bears, cattle, poultry). UDCA can also be produced via a costly chemical synthesis route from cholic acid that has 5-steps including a Wolff-Kishner ketone reduction, and an epimerization at C7 to produce UDCA. See e.g., Eggert, et al., Biotechnol.2014, 191, 11-21. A shorter synthesis route based on the biocatalytic epimerization of chenodeoxycholic acid (CDCA) to UDCA has also been reported. See e.g., Zheng, et al., Process Biochem.2015, 50, 598-604.Biosynthesis of UDCA from lithocholic acid (LCA) using an engineered cytochrome P450 monooxygenase from Streptomyces antibioticus. has been reported in Grobe, et al., “Engineering Regioselectivity of a P450 Monooxygenase Enables the Synthesis of Ursodeoxycholic Acid via 7β-Hydroxylation of Lithocholic Acid,” Angew. Chem. Int. Ed., 60 (2): 753-757 (January 11, 2021); Pages 753-75710.1002 / anie.202012675. The engineered CYP converts LCA to UDCA preferentially over MDCA (73%:27%), but the bioconversion has very ‐ 1 ‐   low productivity ~ 67 mM in 24 hours. WO2022115710A1 describes a recombinant yeast transformed with a naturally occurring cytochrome P450 monooxygenase from Gibberella zeae that is capable of carrying out the biosynthesis of UDCA from LCA, or the structurally related bioconversion of 3-KCA to 3-KUDCA.

[0005] There remains a need for a more efficient recombinant cell systems for the commercially viable biosynthetic production UDCA. SUMMARY

[0006] The present disclosure relates generally to recombinant polypeptides with cytochrome P450 (CYP) activity capable of selectively catalyzing the conversion of precursor compounds, LCA or 3-KCA, to the product compounds, UDCA or 3-KUDCA, and the engineering of these polypeptides for heterologous expression in yeast allowing for improved fermentative bioproduction of these product compounds. This summary is intended to introduce the subject matter of the present disclosure, but does not cover each and every embodiment, combination, or variation that is contemplated and described within the present disclosure. Further embodiments are contemplated and described by the disclosure of the detailed description, drawings, and claims.

[0007] In at least one embodiment, the present disclosure provides a recombinant host cell comprising a heterologous nucleic acid encoding a polypeptide with CYP activity capable of converting LCA to UDCA and / or converting 3-KCA to 3-KUDCA, wherein the polypeptide with CYP activity comprises an amino acid sequence at least 90% sequence identity to SEQ ID NO: 2 and an amino acid difference relative to SEQ ID NO: 2 at one or more positions selected from L7, S31, Q54, V56, K70, K76, I120, A143, L147, A153, N160, H170, G175, E176, V210, V261, T307, N316, Q320, G325, T333, E338, V364, L365, P401, E402, S408, Q424, L444, F452, K479, and M485. In at least one embodiment, the amino acid differences are selected from L7R, S31V, Q54K, V56G, K70L, K70N, K76H, I120A, A143G, A143R, L147R, L147W, A153Q, A153R, N160H, H170V, G175E, E176D, V210R, V261E, T307A, N316S, Q320M, G325E, T333V, E338Q, V364L, L365A, P401D, E402V, S408R, Q424V, L444F, F452D, K479R, and M485T.

[0008] In at least one embodiment of the recombinant host cell, the polypeptide amino acid sequence comprises a combination of amino acid differences selected from: S31V, Q54K, A143G, T307A, G325E‐   ‐   S31V, A143G, G175E, P401D, K498R S31V, A143G, L147C, G175E, P401D 1V P12 A14 R

[0009] In at least one embodiment of the recombinant host cell, the heterologous nucleic acid encoding the polypeptide further comprises a silent mutation selected from R328 (CGT > CGC), A368 (GCT > GCC), and V476 (GTT > GTC).

[0010] In at least one embodiment of the recombinant host cell, the heterologous nucleic acid comprises: (a) a sequence of at least 80% identity to a sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 1185, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, ‐ 3 ‐   1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267, 1269, 1271, and 1275; or (b) a codon degenerate sequence of a sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 1185, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267, 1269, 1271, and 1275.

[0011] In at least one embodiment of the recombinant host cell, the polypeptide with CYP activity comprises an amino acid sequence having at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 2.

[0012] In at least one embodiment, the polypeptide with CYP activity comprises an amino acid sequence selected from SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1244, 1246, 1248, 1250, 1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, and 1272; optionally, wherein the polypeptide is truncated by 1-31 amino acids at its N-terminus.

[0013] In at least one embodiment of the recombinant host cell, the heterologous nucleic acid encodes a polypeptide with CPR activity; optionally, wherein the polypeptide with CPR activity comprises an amino acid sequence of at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 4 and 1278.

[0014] In at least one embodiment of the recombinant host cell, the heterologous nucleic acid encodes a polypeptide with CYP activity capable of converting LCA to UDCA and / or converting 3-KCA to 3-KUDCA fused via a linker to a second polypeptide second polypeptide with CPR activity; optionally, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 1280

[0015] In at least one embodiment of the recombinant host cell, the heterologous nucleic acid is integrated into a site in the host cell genome. In at least one embodiment, the heterologous nucleic acid is integrated at one or more sites in the genome; optionally, integrated at three or more sites. In at least one embodiment, the heterologous nucleic acid is under the control of a promoter system selected from pGal1 / 10, and pCAT1:pFDH1.

[0016] In at least one embodiment of the recombinant host cell, the source organism of the recombinant host cell is selected from Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, and Escherichia coli. In at least one embodiment, the recombinant host cell is ‐ 4 ‐   Saccharomyces cerevisiae and the heterologous nucleic acid is integrated at one or more sites in the genome selected from X-4, XI-2, XII-4, and X-2. In at least one embodiment, the heterologous nucleic acid is integrated at three or more sites; optionally, integrated at four or more sites.

[0017] In at least one embodiment of the recombinant host cell, the recombinant host cell is Pichia pastoris and the heterologous nucleic acid is integrated at one or more sites in the genome selected from AOX1, Int6, Int15, and HIS4. In at least one embodiment, the heterologous nucleic acid is integrated at three or more sites; optionally, integrated at four or more sites.

[0018] In at least one embodiment, the present disclosure also provides a method for producing UDCA or 3-KUDCA comprising: (a) culturing a recombinant host cell of the present disclosure in a suitable medium comprising LCA and / or 3-KCA; and (b) recovering the produced UDCA and / or 3-KUDCA.

[0019] In at least one embodiment, the present disclosure also provides a recombinant polypeptide with CYP activity capable of converting LCA to UDCA and / or converting 3-KCA to 3-KUDCA, wherein the polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 and an amino acid difference relative to SEQ ID NO: 2 at one or more positions selected from L7, S31, Q54, V56, K70, K76, I120, A143, L147, A153, N160, H170, G175, E176, V210, V261, T307, N316, Q320, G325, T333, E338, V364, L365, P401, E402, S408, Q424, L444, F452, K479, and M485. In at least one embodiment, the amino acid differences are selected from L7R, S31V, Q54K, V56G, K70L, K70N, K76H, I120A, A143G, A143R, L147R, L147W, A153Q, A153R, N160H, H170V, G175E, E176D, V210R, V261E, T307A, N316S, Q320M, G325E, T333V, E338Q, V364L, L365A, P401D, E402V, S408R, Q424V, L444F, F452D, K479R, and M485T. In at least one embodiment, the polypeptide amino acid sequence comprises a combination of amino acid differences selected from: S31V, Q54K, A143G, T307A, G325E S31V Q54K A143G T307A G325E L365A‐ 5 ‐   S31V, Q54K, A143G, A153Q, T307A, G325E S31V, Q54K, A143G, A153P, T307A, G325E S31V Q54K A143G T307A G325E Q424V [p yp p , p yp p p o acid sequence having at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 2.

[0021] In at least one embodiment, the polypeptide comprises an amino acid sequence selected from SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1244, 1246, 1248, 1250, 1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, 1272, and 1276.

[0022] In at least one embodiment, the recombinant polypeptide with CYP activity capable of converting LCA to UDCA and / or converting 3-KCA to 3-KUDCA of the present disclosure comprises an N-terminal truncation of from 1 to 31 amino acids as compared to SEQ ID NO: 2. In at least one embodiment, the polypeptide comprises an N-terminal truncation of from 1 to 31 amino acids of an amino acid sequence selected from SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 1186, 1188, 1190, ‐ 6 ‐   1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1244, 1246, 1248, 1250, 1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, and 1272. In at least one embodiment, the polypeptide comprises an amino acid of SEQ ID NO: 1276.

[0023] In at least one embodiment, the recombinant polypeptide with CYP activity capable of converting LCA to UDCA and / or converting 3-KCA to 3-KUDCA of the present disclosure is fused via a linker to a second polypeptide. In at least one embodiment, the second polypeptide has CPR activity. In at least one embodiment, the second polypeptide with CPR activity comprises an amino acid sequence having at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 4 and 1278.

[0024] In at least one embodiment, the recombinant polypeptide with CYP activity capable of converting LCA to UDCA and / or converting 3-KCA to 3-KUDCA is fused via a linker to a second polypeptide second polypeptide with CPR activity; optionally, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 1280.

[0025] In at least one embodiment, the present disclosure also provides a polynucleotide encoding a polypeptide of the present disclosure. In at least one embodiment, the polynucleotide sequence comprises: (a) a sequence of at least 80% identity to a sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 1185, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267, 1269, 1271, and 1275; or (b) a codon degenerate sequence of a sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 1185, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267, 1269, 1271, and 1275.

[0026] In at least one embodiment, the present disclosure also provides an expression vector comprising a polynucleotide of the present disclosure. In at least one embodiment, the expression vector comprises a control sequence.

[0027] In at least one embodiment, the present disclosure also provides a host cell comprising a polynucleotide or an expression vector of the present disclosure.

[0028] In another embodiment, the present disclosure provides a method for preparing a compound of structural formula (I) ‐ 7 ‐   wherein, R1is hydrogen, substituted C1-C20 alkyl, or an optionallyis a hydroxyl, or an oxo group; the method comprising contacting under suitable reactions conditions a compound of structural formula (II) wherein, R1is substituted C1-C20 alkyl,or an optionally or a is a hydroxyl, or an oxo group; and a recombinant polypeptide of the present disclosure.

[0029] In at least one embodiment of the method for preparing a compound of structural formula (I), R2is hydrogen, and (a) the compound of structure formula (I) is UDCA and the compound of structural formula (II) is LCA; or (b) the compound of structure formula (I) is 3- KUDCA and the compound of structural formula (II) is 3-KCA.

[0030] In at least one embodiment of the method for preparing a compound of structural formula (I), R2is a hydroxyl, and the compound of structure formula (I) is UCA and the compound of structural formula (II) is DCA. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] A better understanding of the novel features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0032] FIG.1 depicts Schemes A and B used for PCR primer synthesis of DNA fragments used in building strains of Saccharomyces cerevisiae with CPR and CYP genes under control of pGal1 / 10 bidirectional promoter as described in Example 1. ‐ 8 ‐

[0033] FIG.2 depicts the lineage of the four Saccharomyces cerevisiae strains (SH013, SH021, SH020, and SHP025) built as described in Example 1.

[0034] FIG.3 depicts lineage of Pichia pastoris strains built as described in Example 4.

[0035] FIG.4 depicts lineage of Pichia pastoris strains built as described in Examples 7-9.

[0036] FIG.5 depicts a plot of screening results for Pichia pastoris strains built as described in Example 8.

[0037] FIG.6 depicts plots of screening results for Pichia pastoris strains built as described in Example 9. DETAILED DESCRIPTION

[0038] For the descriptions herein and the appended claims, the singular forms “a”, and “an” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a protein” includes more than one protein, and reference to “a compound” refers to more than one compound. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. The use of “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”

[0039] Where a range of values is provided, unless the context clearly dictates otherwise, it is understood that each intervening integer of the value, and each tenth of each intervening integer of the value, unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of these limits, ranges excluding (i) either or (ii) both of those included limits are also included in the invention. For example, “1 to 50,” includes “2 to 25,” “5 to 20,” “25 to 50,” “1 to 10,” etc.

[0040] Generally, the nomenclature used herein and the techniques and procedures described herein include those that are well understood and commonly employed by those of ordinary skill in the art, such as the common techniques and methodologies described in e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Vols.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2012 (hereinafter “Sambrook”); and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., originally published in 1987 in book ‐ 9 ‐   form by Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., and regularly supplemented through 2011, and now available in journal format online as Current Protocols in Molecular Biology, Vols.00 - 130, (1987-2020), published by Wiley & Sons, Inc. in the Wiley Online Library (hereinafter “Ausubel”).

[0041] All publications, patents, patent applications, and other documents referenced in this disclosure are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference herein for all purposes.

[0042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. It is to be understood that the terminology used herein is for describing particular embodiments only and is not intended to be limiting. For purposes of interpreting this disclosure, the following description of terms will apply and, where appropriate, a term used in the singular form will also include the plural form and vice versa.

[0043] Definitions

[0044] “UDCA” refers to the compound ursodeoxycholic acid having the chemical structure shown as compound 1a in Table 1 (below).

[0045] “UDCA derivatives” as referenced in the present disclosure include, but are not limited to, structural analogs of UDCA, such as 7β-hydroxy-3-oxo-5β-cholanoic acid (3-KUDCA), compound 1b, and the other exemplary compounds shown below in Table 1 (below).

[0046] TABLE 1: Exemplary UDCA and UDCA derivative compounds  Abbrev. Compound Name Name Chemical Structure‐ 10 ‐   Methyl Ursodeoxycholate Me-UDCA

[0047] “UDCA precursor” as used herein refers to a compound capable of being converted into UDCA, or a UDCA derivative, by an enzyme capable producing UDCA alone, or in combination with another enzyme or a non-enzymatic chemical reaction. UDCA precursors as referenced in the present disclosure include, but are not limited to, the exemplary compounds summarized in Table 2 (below).

[0048] TABLE 2: Exemplary UDCA and UDCA derivative precursor compounds  Abbrev.‐ 11 ‐   Lithocholic acid LCA[ ] ac y as use ee ee s o e caay c ac y o a cyoc o e monooxygenase enzyme.

[0050] “CPR activity” as used herein refers to the catalytic activity of a cytochrome P450 reductase enzyme.

[0051] “Conversion” as used herein refers to the enzymatic conversion of a substrate(s) to a corresponding product(s). “Percent conversion” refers to the percent of the substrate that is converted to the product within a period of time under specified conditions. Thus, the “enzymatic activity” or “activity” of an enzymatic conversion can be expressed as “percent conversion” of the substrate to the product.

[0052] “Substrate” as used herein in the context of an enzyme mediated process refers to the compound or molecule acted on by the enzyme.

[0053] “Product” as used herein in the context of an enzyme mediated process refers to the compound or molecule resulting from the activity of the enzyme.

[0054] “Host cell” as used herein refers to a cell capable of being functionally modified with recombinant nucleic acids and functioning to express recombinant products, including polypeptides and compounds produced by activity of the polypeptides. ‐ 12 ‐

[0055] “Nucleic acid,” or “polynucleotide” as used herein interchangeably to refer to two or more nucleosides that are covalently linked together. The nucleic acid may be wholly comprised ribonucleosides (e.g., RNA), wholly comprised of 2'-deoxyribonucleotides (e.g., DNA) or mixtures of ribo- and 2'-deoxyribonucleosides. The nucleoside units of the nucleic acid can be linked together via phosphodiester linkages (e.g., as in naturally occurring nucleic acids), or the nucleic acid can include one or more non-natural linkages (e.g., phosphorothioester linkage). Nucleic acid or polynucleotide is intended to include single- stranded or double-stranded molecules, or molecules having both single-stranded regions and double-stranded regions. Nucleic acid or polynucleotide is intended to include molecules composed of the naturally occurring nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), or molecules comprising that include one or more modified and / or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc.

[0056] “Protein,” “polypeptide,” and “peptide” are used herein interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristilation, ubiquitination, etc.). As used herein “protein” or “polypeptide” or “peptide” polymer can include D- and L-amino acids, and mixtures of D- and L-amino acids.

[0057] “Naturally-occurring” or “wild-type” as used herein refers to the form as found in nature. For example, a naturally occurring nucleic acid sequence is the sequence present in an organism that can be isolated from a source in nature, and which has not been intentionally modified by human manipulation.

[0058] “Recombinant,” “engineered,” or “non-naturally occurring” when used herein 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, or is identical thereto but is produced or derived from synthetic materials and / or by manipulation using recombinant techniques. 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.

[0059] “Nucleic acid derived from” as used herein refers to a nucleic acid having a sequence at least substantially identical to a sequence of found in naturally in an organism. For example, cDNA molecules prepared by reverse transcription of mRNA isolated from an organism, or nucleic acid molecules prepared synthetically to have a sequence at least substantially identical to, or which hybridizes to a sequence at least substantially identical to a nucleic sequence found in an organism.

[0060] “Coding sequence” refers to that portion of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein. ‐ 13 ‐

[0061] “Heterologous nucleic acid” as used herein refers to any polynucleotide that is introduced into a host cell by laboratory techniques and includes polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into a host cell.

[0062] “Codon degenerate” describes a nucleotide sequence that has one or more different codons relative to the reference nucleotide sequence, but which encodes a polypeptide that is identical to the polypeptide encoded by a reference nucleotide sequence. The different codons between the nucleotide sequence and the reference nucleotide sequence are called “synonyms” or “synonymous” codons in that they use different triplets of nucleotides to encode the same amino acid in a polypeptide.

[0063] “Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several different “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the imine reductase enzymes may be codon optimized for optimal production from the host organism selected for expression.

[0064] “Preferred, optimal, high codon usage bias codons” refers to codons that are used at higher frequency in the protein coding regions than other codons that code for the same amino acid. The preferred codons may be determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, the codon frequency in the aggregate protein coding regions of the whole organism, codon frequency in the aggregate protein coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression. A variety of methods are known for determining the codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in specific organisms, including multivariate analysis, for example, using cluster analysis or correspondence analysis, and the effective number of codons used in a gene (see GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, John Peden, University of Nottingham; McInerney, J. O, 1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res.222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables are available for a growing list of organisms (see for example, Wada et al., 1992, Nucleic Acids Res.20:2111-2118; Nakamura et al., 2000, Nucl. Acids Res.28:292; Duret, et al., supra; Henaut and Danchin, "Escherichia coli and Salmonella," 1996, Neidhardt, et al. Eds., ASM Press, Washington D.C., p.2047-2066. The data source for obtaining codon usage may rely on any available nucleotide sequence capable of coding for a ‐ 14 ‐   protein. These data sets include nucleic acid sequences actually known to encode expressed proteins (e.g., complete protein coding sequences-CDS), expressed sequence tags (ESTS), or predicted coding regions of genomic sequences (see for example, Mount, D., Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Uberbacher, E. C., 1996, Methods Enzymol.266:259-281; Tiwari et al., 1997, Comput. Appl. Biosci.13:263-270).

[0065] “Control sequence” as used herein refers to all sequences, which are necessary or advantageous for the expression of a polynucleotide and / or polypeptide as used in the present disclosure. Each control sequence may be native or foreign to the nucleic acid sequence encoding a polypeptide. Such control sequences include, but are not limited to, a leader, a promoter, a polyadenylation sequence, a pro-peptide sequence, a signal peptide sequence, and a transcription terminator. At a minimum, control sequences typically include a promoter, and transcriptional and translational stop signals. 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.

[0066] “Operably linked” as used herein refers to a configuration in which a control sequence is appropriately placed (e.g., in a functional relationship) at a position relative to a polynucleotide sequence or polypeptide sequence of interest such that the control sequence directs or regulates the expression of the sequence of interest.

[0067] “Promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of a polynucleotide of interest. 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.

[0068] “Percentage of sequence identity,” “percent sequence identity,” “percentage homology,” or “percent homology” are used interchangeably herein to refer to values quantifying comparisons of the sequences of polynucleotides or 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 may comprise additions or deletions (or gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage values may be 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. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino ‐ 15 ‐   acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap 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. Those of skill in the art 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, a joint 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. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). Exemplary determination of sequence alignment and % sequence identity can ‐ 16 ‐   employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison Wis.), using default parameters provided.

[0069] “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 nucleic acid or polypeptide sequence. A reference sequence typically is at least 20 nucleotide or amino acid residue units in length but can also be 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 or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity. “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 (or 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.

[0070] “Substantial identity” or “substantially identical” refers to a polynucleotide or polypeptide sequence that has at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95 % sequence identity, or at least 99% sequence identity, as compared to a reference sequence over a comparison window of at least 20 nucleoside or amino acid residue positions, frequently over a window of at least 30-50 positions, wherein the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that includes deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.

[0071] “Corresponding to,” “reference to,” or “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of an engineered imine reductase, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned. ‐ 17 ‐

[0072] “Isolated” as used herein in reference to a molecule means that the molecule (e.g., UDCA, polynucleotide, polypeptide) is substantially separated from other compounds that naturally accompany it, e.g., protein, lipids, and polynucleotides. The term embraces nucleic acids which have been removed or purified from their naturally occurring environment or expression system (e.g., host cell or in vitro synthesis).

[0073] “Substantially pure” refers to a composition in which a desired molecule 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.

[0074] “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.

[0075] Engineered Genes Encoding Recombinant Polypeptides with CYP Activity

[0076] The present disclosure provides engineered genes encoding a polypeptide with cytochrome P450 (CYP) monooxygenase activity capable of converting LCA to UDCA and / or capable of converting 3-KCA to 3-KUDCA. The engineered CYP genes are derived from a naturally occurring parent CYP gene isolated from Gibberella zeae that encodes the 513 amino acid CYP polypeptide of SEQ ID NO: 2. MATDLDLVLGKSQYALFCGITLFSFFILKYSLLGNGGKQYPYINPKKPFELSNQRVVQDFIENARDILTK GRSLYKDTPYKAHTDLGDVLVIPPEFADALKSERQLDFTEVARDDTHGYIPGFEPIGSPFDLVPLVNKYL TRALAKLTKPLWAEASLGVNHVLGTSTEWHPINPGEDIMRIVSRMSSRIFMGEELCKDDDWLKVSIEYTV QLFQTADELRNYPRWTRPYIHWFLPSCQGVRRKLQEARDLLQPHIDRRNAVKKEAIAEGRPSPFDDSIEW FENEYEGKSDPATEQIKLSLVAIHTTTDLLSETMFNIALQPELLGPLREEIVTVLSTEGLKKTSFYNLKL MDSVIKESQRLRPVLLGAFRRMALADVTLPNGDVIKKGTKIICDTTHQWNPEYYPDASKFNAYRFLQMRQ TPGQDKRAHLVSTSHDQMGFGHGLHACPGRFFAANEIKIALCHMLLKYDWKLPEGVVPKSKALGMSLLGD REAKLMVKRRAAEIDIDTIGSDE (SEQ ID NO: 2)

[0077] In at least one embodiment of the present disclosure, the 513 amino acid CYP polypeptide of SEQ ID NO: 2 can be encoded by the yeast codon-optimized 1542 nucleotide sequence of SEQ ID NO: 1. ATGGCCACCGATCTAGACCTAGTATTAGGAAAAAGTCAATACGCATTATTTTGTGGCATAACTTTATTTA GCTTTTTCATACTAAAGTATAGTCTTCTCGGAAACGGGGGCAAGCAATATCCTTATATCAACCCTAAGAA ACCTTTTGAGCTGTCGAATCAACGAGTTGTCCAGGATTTTATCGAGAACGCACGAGACATTTTGACTAAG GGTCGCTCACTTTACAAGGATACGCCCTACAAGGCTCATACCGATTTAGGGGACGTCTTGGTAATCCCGC CTGAATTTGCCGACGCTCTAAAATCTGAAAGACAGTTAGATTTTACCGAAGTCGCAAGAGACGATACTCA CGGTTATATTCCTGGATTCGAGCCAATCGGTTCCCCGTTCGATCTGGTGCCGCTCGTTAACAAGTATCTT ACAAGGGCTTTGGCAAAACTAACAAAACCATTGTGGGCCGAAGCTTCTTTAGGTGTAAACCATGTTTTAG GCACTTCTACTGAGTGGCATCCTATTAACCCAGGGGAAGATATCATGAGGATTGTCTCCAGAATGTCATC CAGAATATTCATGGGTGAGGAACTTTGTAAAGATGATGATTGGTTGAAAGTTAGTATTGAGTACACTGTG CAATTGTTTCAAACCGCAGACGAATTACGTAACTATCCACGTTGGACAAGACCATATATTCACTGGTTTT TGCCTTCCTGTCAAGGGGTTAGGAGAAAATTGCAGGAAGCGCGTGATTTATTGCAACCCCATATTGATAG GAGAAATGCTGTGAAGAAAGAAGCTATCGCTGAAGGTAGACCATCACCATTCGACGATTCAATAGAATGG TTTGAAAATGAATACGAGGGCAAATCTGATCCCGCCACTGAACAAATTAAACTATCACTGGTGGCGATTC ACACAACAACTGATTTACTGTCTGAAACAATGTTTAATATAGCTTTGCAACCAGAACTCCTTGGTCCACT ACGTGAAGAGATAGTTACGGTTTTATCCACGGAAGGTCTAAAAAAGACGTCGTTTTACAATTTGAAGTTG ‐ 18 ‐   ATGGATTCGGTCATAAAAGAGTCACAGAGACTTCGCCCTGTTTTATTAGGTGCTTTCAGGAGAATGGCAT TGGCTGATGTTACCTTGCCCAATGGCGACGTAATTAAAAAAGGTACCAAGATCATTTGCGACACTACACA TCAATGGAATCCAGAATACTATCCAGATGCTAGTAAGTTCAATGCATATAGATTTTTGCAAATGAGACAG ACACCGGGTCAGGACAAAAGAGCACACCTTGTCAGCACAAGCCATGATCAAATGGGATTCGGACATGGCT TGCACGCGTGCCCAGGAAGATTTTTCGCAGCCAATGAGATTAAGATTGCGCTGTGTCATATGCTATTGAA ATATGATTGGAAATTACCAGAAGGTGTTGTACCTAAGTCTAAGGCTTTAGGAATGTCTTTACTTGGTGAC CGGGAAGCCAAACTGATGGTGAAAAGGAGAGCAGCCGAAATCGATATAGACACTATTGGTAGTGATGAAT AA (SEQ ID NO: 1)

[0078] As described elsewhere herein, the polynucleotide sequence of SEQ ID NO: 1, or alternative codon-optimized sequences that encode the CYP polypeptide of SEQ ID NO: 2, can be used as the parent gene sequence for engineering of the CYP polypeptide for production of UDCA in yeast.

[0079] In the presence of a cytochrome P450 reductase (CPR) enzyme, the cofactor NADPH, and oxygen, the 7-position hydroxylating monooxygenase activity of the recombinant CYP polypeptides encoded by the engineered genes of the present disclosure is capable of catalyzing the conversion of LCA (compound 2a) to UDCA (compound 1a) as shown by the upper reaction illustrated in Scheme 1 (below). Scheme 1

[0080] Similarly, the CYP activity of the polypeptides encoded by the engineered genes of the present disclosure is capable of catalyzing the conversion of 3-KCA (compound 2b) to 3- KUDCA (compound 1b) as shown by the lower reaction illustrated in Scheme 1. The 3-KUDCA product can then be converted to UDCA by a further keto reduction reaction carried out by a ketoreductase (KRED) enzyme.

[0081] Additionally, the CYP activity of the polypeptides encoded by the engineered genes of the present disclosure is capable of catalyzing the 7-position hydroxylation of DCA (compound ‐ 19 ‐   2c) to UCA (compound 1e) and / or CA (compound 1f) as shown by the reaction illustrated in Scheme 2. Scheme 2DCA to UCA / CA conversion of Scheme 2) can be provided by the 264 amino acid CPR from Gibberella zeae of SEQ ID NO: 4. MALRTSLSRPVPLLATLTASAIGVSILSKMMFSTASAESPSPQKIFSGAFASVKLPLHSSEYESHDTKRL RFKLPQETAVTGLPLAYLVHIPPSHHQRDLTTPDEPGYMDLLVKKYPKGQGSTYLHSLQPGDTLSFTSLP LKPAWKTNNFPHITLIAGGCGITPLFNLAQGILRDPAEKTRMTFIFGARSDEDVLLKKELDGFAKEFPER FEVKYTALLEEVLGGVGRDTKVFVCGPKEMEKALVGGRGVLKEIGFEKSQIHTF (SEQ ID NO: 4)

[0083] In at least one embodiment of the present disclosure, the 264 amino acid CPR polypeptide of SEQ ID NO: 4 can be encoded by a yeast codon-optimized 795 nucleotide sequence of SEQ ID NO: 3. ATGGCACTACGAACATCACTCTCTCGCCCCGTTCCCTTGTTAGCTACCCTGACTGCCAGCGCAATCGGAG TATCTATATTGTCTAAAATGATGTTTTCAACCGCCAGTGCCGAGAGTCCATCTCCGCAAAAAATTTTTTC CGGTGCTTTTGCTTCCGTAAAACTACCGCTGCATTCAAGTGAATACGAATCCCATGATACAAAGAGGTTG CGTTTCAAACTTCCTCAAGAGACTGCAGTCACGGGTTTACCATTAGCTTACTTGGTTCACATTCCACCTA GCCACCATCAAAGAGACTTGACTACGCCGGATGAACCTGGATACATGGACCTGTTGGTGAAGAAATATCC CAAAGGTCAGGGTTCGACATATCTACACTCGCTCCAGCCAGGTGATACCTTATCATTCACATCTCTACCA TTGAAACCAGCGTGGAAAACAAATAATTTTCCTCATATCACTCTTATTGCTGGAGGGTGTGGGATTACGC CATTATTCAACTTGGCTCAAGGTATACTTAGAGATCCTGCCGAAAAAACTAGGATGACCTTTATTTTTGG TGCAAGATCAGACGAGGATGTTTTACTGAAAAAGGAGTTAGATGGCTTTGCAAAAGAGTTCCCAGAAAGA TTCGAAGTGAAATATACAGCGCTTTTGGAAGAAGTCCTAGGAGGCGTGGGTCGTGATACTAAGGTTTTTG TCTGCGGTCCTAAGGAAATGGAAAAGGCTTTAGTTGGAGGCAGAGGTGTATTAAAGGAAATAGGCTTCGA AAAGTCTCAAATCCATACTTTTTAA (SEQ ID NO: 3)

[0084] As described elsewhere herein, the polynucleotide of SEQ ID NO: 3, or an alternative codon-optimized polynucleotide that encodes a CPR polypeptide of SEQ ID NO: 4, can be used in a heterologous nucleic acid along with a gene (such as SEQ ID NO: 1) encoding the CYP polypeptide of SEQ ID NO: 2 to generate a recombinant host cell capable of carrying out the biocatalytic conversion of LCA to UDCA and / or 3-KCA to 3-KUDCA

[0085] WO2022115710A1 describes a yeast strain denoted “SAND122” that is transformed with a gene encoding the CYP of SEQ ID NO: 2 and a gene encoding the CPR of SEQ ID NO: 4. The SAND122 strain is capable of converting LCA to UDCA and / or converting 3-KCA to 3- KUDCA.

[0086] In at least one embodiment, when a host cell (e.g., S. cerevisiae or P. pastoris) is transformed with a heterologous nucleic acid comprising an engineered CYP gene of the present disclosure, a gene encoding a CPR polypeptide (e.g., SEQ ID NO: 4), and the ‐ 20 ‐   recombinant host cell is fed LCA, the product UDCA, is produced by the host cell in greater yield relative to a comparable recombinant host cell integrated with the parent gene encoding the wild-type CYP polypeptide of SEQ ID NO: 2. Without intending to be bound by any particular theory or mechanism, the enhanced yield of the UDCA and / or 3-KUDCA biosynthetic product is correlated with the one or more amino acid residue differences in recombinant polypeptides of the present disclosure, as compared to the amino acid sequence of wild-type CYP from G. zeae of SEQ ID NO: 2 from which the engineered polypeptide sequences are derived.

[0087] Exemplary engineered CYP genes and encoded recombinant polypeptides with CYP activity that exhibit the unexpected and surprising technical effect of comparable or increased UDCA yield when integrated in a recombinant host cell are summarized in Table 3 below (as well as in the following Examples and the accompanying Sequence Listing).

[0088] TABLE 3: Engineered CYP polypeptides Silent NT AA d n SEQ SEQ :‐ 21 ‐ TEVARDDTHGYIPGFEPIGSPFDLVPLVNKYLTRAL AKWTKPLWAEASLGVNHVLGTSTEWHPINPGEDIMR IVSRMSSRIFMGEELCKDDDWLKVSIEYTVQLFQTA‐ 22 ‐ MATDLDLVLGKSQYALFCGITLFSFFILKYSLLGNG Q54K 17 18 GKQYPYINPKKPFELSNKRVVQDFIENARDILTKGR SLYKDTPYKAHTDLGDVLVIPPEFADALKSERQLDF    DWKLPEGVVPKSKALGMSLLGDREAKLMVKRRAAEI DIDTIGSDE* MATDLDLVLGKSQYALFCGITLFSFFILKYSLLGNG K76H 25 26      HQWNPEYYPDASKFNAYRFLQMRQTPGQDKRAHLVS TSHDQMGFGHGLHACPGRFFAANEIKIALCHMLLKY DWKLPEGVVPKSKALGMSLLGDREAKLMVKRRAAEI      GPLREEIVTVLSTEGLKKTSFYNLKLMDSVIKESQR LRPVLLGAFRRMALADVTLPNGDVIKKGTKIICDTT HQWNDEYYPDASKFNAYRFLQMRQTPGQDKRAHLVS      PHIDRRNAVKKEAIAEGRPSPFDDSIEWFENEYEGK SDPATEQIKLSLVAIHTTTDLLSETMFNIALQPELL GPLREEIVTVLSTEGLKKTSFYNLKLMDSVIKESQR    IVSRMSSRIFMGEELCKDDDWLKVSIEYTVQLFQTA DELRNYPRWTRPYIHWFLPSCQGVRRKLQEARDLLQ PHIDRRNAVKKEAIAEGRPSPFDDSIEWFENEYEGK 6 8‐ 28 ‐ MATDLDRVLGKSQYALFCGITLFSFFILKYVLLGNG L7R, S31V, 1189 1190 GKQYPYINPKKPFELSNKRVVQDFIENARDILTKGR Q54K, A143G, SLYKDTPYKAHTDLGDVLVIPPEFADALKSERQLDF T307A, G325E 2 4 6     DWKLPEGVVPKSKALGMSLLGDREAKLMVKRRAAEI DIDTIGSDE MATDLDLVLGKSQYALFCGITLFSFFILKYVLLGNG S31V, Q54K, 1197 1198 0 2 4    HQWNPEYYPDASKFNAYRFLQMRQTPGQDKRAHLVS TSHDQMGFGHGLHACPGRFFAANEIKIALCHMLLKY DWKLPEGVVPKSKALGMSLLGDREAKLMVKRRAAEI 6 8 0 2    EPLREEIVTVLSTEGLKKTSFYNLKLMDSVIKESQR LRPVLLGAFRRMALADVTLPNGDVIKKGTKIICDTT HQWNPEYYPDASKFNAYRFLQMRQTPGQDKRAHLVS 4 6 8 0    PHIDRRNAVKKEAIAEGRPSPFDDSIEWFENEYEGK SDPATEQIKLSLVAIHTTADLLSETMFNIALQPELL EPLREEIVTVLSTEGLKKTSFYNLKLMDSVIKESQR 4 6 8 0    IVSRMSSRIFMGEELCKDDDWLKVSIEYTVQLFQTA DELRNYPRWTRPYIHWFLPSCQGVRRKLQEARDLLQ PHIDRRNAVKKEAIAEGRPSPFDDSIEWFENEYEGK 2 4 6 8    TEVARDDTHGYIPGFEPIGSPFDLVPLVNKYLTRGL V364L, E338Q, AKLTKPLWAEASLGVNHVLGTSTEWHPINPGEDIMR M485T IVSRMSSRIFMGEELCKDDDWLKVSIEYTVQLFQTA 0 2 4‐ 35 ‐ MATDLDRVLGKSQYALFCGITLFSFFILKYVLLGNG L7R, S31V, 1265 1266 GKQYPYINPKKPFELSNKRVVQDFIENARDILTLGR Q54K, K70L, SLYKDTPYKAHTDLGDVLVIPPEFADALKSERQLDF A143G, H170V, 8 0 2       DWKLPEGVVPKSKALGMSLLGDREAKLMVKRRAAEI DIDTIGSDE MLGNGGKQYPYINPKKPFELSNKRVVQDFIENARDI 31-aa N-term 1275 1276 :, ne or more residue differences as compared to the reference G. zeae CYP polypeptide of SEQ ID NO: 2. In some embodiments, the recombinant polypeptides have one or more amino acid residue differences as compared to SEQ ID NO: 2 at an amino acid position selected from L7, S31, Q54, V56, K70, K76, I120, A143, L147, A153, N160, H170, G175, E176, V210, V261, T307, N316, Q320, G325, T333, E338, V364, L365, P401, E402, S408, Q424, L444, F452, K479, and M485. In some embodiments, the recombinant polypeptides have one or more amino acid residue differences as compared to SEQ ID NO: 2 selected from L7R, S31V, Q54K, V56G, K70L, K70N, K76H, I120A, A143G, A143R, L147R, L147W, A153Q, A153R, N160H, H170V, G175E, E176D, V210R, V261E, T307A, N316S, Q320M, G325E, T333V, E338Q, V364L, L365A, P401D, E402V, S408R, Q424V, L444F, F452D, K479R, and M485T.

[0090] It is contemplated that the residue differences relative to SEQ ID NO: 2 at residue positions associated with increased CYP activity can be used in various combinations to form recombinant CYP polypeptides having desirable functional characteristics when integrated in a recombinant host cell, for example increased yield of a product compound, such as UDCA. Some exemplary combinations of amino acid differences include those combinations found in the exemplary polypeptides of Table 3 and elsewhere herein. For example, the present disclosure provides a recombinant polypeptide having increased CYP activity, a set of amino acid residue differences as compared to SEQ ID NO: 2 selected from: S31V, Q54K, A143G, T307A, G325E‐ 37 ‐   S31V, Q54K, A143R, L147R, T307A, G325E, P401D S31V, V57F, L147W, P401D S31V L147W P401D

[0091] Based on the correlation of recombinant polypeptide functional information provided herein with the sequence information provided in Table 3, the accompanying Sequence Listing, and / or the Examples disclosed herein, one of ordinary skill can recognize that the present disclosure provides a range of recombinant polypeptides having CYP activity, wherein the polypeptide comprises an amino acid sequence comprising one or more of the amino acid differences or sets of amino acid differences (relative to SEQ ID NO: 2) disclosed in any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, ‐ 38 ‐   50, 52, 54, 56, 58, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1244, 1246, 1248, 1250, 1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, 1272, and 1276, and otherwise have at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1244, 1246, 1248, 1250, 1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, 1272, and 1276.

[0092] Additionally, in at least one embodiment, a recombinant polypeptide of the present disclosure having CYP activity can have an amino acid sequence comprising one or more of the amino acid differences or sets of amino acid differences (relative to SEQ ID NO: 2) disclosed in any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1244, 1246, 1248, 1250, 1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, 1272, and 1276, and additionally have 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residue differences at other residue positions. In some embodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences at the other residue positions.

[0093] In addition to the residue positions specified above, any of the engineered CYP polypeptides disclosed herein can further comprise other residue differences relative to the reference polypeptide of SEQ ID NO: 2 at other residue positions.

[0094] Residue differences at these other residue positions can provide for additional variations in the amino acid sequence without adversely affecting the ability of the recombinant polypeptide to carry out the desired biocatalytic conversion (e.g., conversion of LCA (compound 2a) to UDCA (compound 1a)). In some embodiments, the recombinant polypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40 residue differences at other amino acid residue positions as compared to SEQ ID NO: 2. In some embodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, and 40 residue differences at other residue positions. The residue difference at these other positions can include conservative changes or non-conservative changes. In some embodiments, the residue differences can comprise conservative substitutions and non-conservative substitutions as compared to the reference polypeptide of SEQ ID NO: 2. ‐ 39 ‐

[0095] Other engineered modifications of the CYP polypeptides contemplated by the present disclosure include modification of the amino acid sequence at either its N- or C- terminus by truncation or fusion. For example, in at least one embodiment, the CYP polypeptides (or CPR polypeptides) of the present disclosure can be further engineered by truncation at the N- and / or C-terminus of the polypeptide chain to provide a truncated version of the polypeptide that retains CYP activity. Methods for preparing and screening truncated versions of CYP polypeptides are known in the art and described in the Examples and elsewhere herein. The present disclosure contemplates that the genes encoding these truncated CYP and CPR enzymes can be used in the recombinant host cell compositions and accompanying biosynthetic methods of the present disclosure. In one embodiment, the engineered CYP polypeptide CYP207 (SEQ ID NO: 1186) of the present disclosure has been prepared in a form that is truncated at the N-terminus by 31 amino acids. This truncated version of CYP207, referred to as trCYP207 (SEQ ID NO: 1276) retains CYP activity a gene encoding it is heterologously expressed in a recombinant Pichia host cell. Accordingly, in at least one embodiment any of the engineered CYP polypeptides disclosed herein can include an engineered version of the polypeptide (and the encoding gene), wherein from 1-31 N-terminal amino acids are truncated.

[0096] In at least one embodiment, such a recombinant polypeptide with an N-terminal truncation and CYP activity capable of converting LCA to UDCA and / or converting 3-KCA to 3- KUDCA comprises an N-terminal truncation of 1 to 31 amino acids as compared to SEQ ID NO: 2. It is further contemplated that the polypeptide with CYP activity can comprise an N-terminal truncation of from 1 to 31 amino acids of engineered recombinant polypeptide comprising a sequence of any of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1244, 1246, 1248, 1250, 1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, and 1272. In at least one embodiment, the N-terminal truncated polypeptide with CYP activity comprises an amino acid of SEQ ID NO: 1276.

[0097] It also is contemplated that the engineered CYP (and CPR) polypeptides of the present disclosure can be modified via fusion to other polypeptides. In at least one embodiment of a fusion, an engineered recombinant gene encoding a polypeptide having CYP activity of the present disclosure is fused to a gene encoding a recombinant polypeptide having CPR activity, whereby the fusion gene when expressed heterologously in a recombinant host cell expression a fusion polypeptide having both CYP activity and CPR activity. As shown in the Examples and elsewhere herein, such engineered CYP-CPR fusion polypeptides can be used in the biosynthetic processes for preparing UDCA.

[0098] In at least one embodiment, the recombinant polypeptide with CYP activity is fused via a polypeptide linker to a second polypeptide with CPR activity. In at least one embodiment of the ‐ 40 ‐   fusion, the polypeptide with CYP activity used in the fusion is an 1-31 amino acid N-terminal truncated version of an engineered polypeptide comprising a sequence of at least 80%, 90%, 95%, or 99% identity to any of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1244, 1246, 1248, 1250, 1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, and 1272. In at least one embodiment of the fusion, the second polypeptide with CPR activity comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to a sequence selected from SEQ ID NO: 4 and 1278.

[0099] In at least one embodiment the recombinant fusion polypeptide with CYP activity and CPR activity is capable of converting LCA to UDCA and / or converting 3-KCA to 3-KUDCA is fused and comprises an amino acid sequence of at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 1280.

[0100] It is further contemplated that the engineered polypeptides of the present disclosure can be fused to polypeptides such as antibody tags (e.g., myc epitope), purification sequences (e.g., His tags for binding to metals), and cell localization signals (e.g., secretion signals). It is also contemplated that the recombinant polypeptides described herein are not restricted to the genetically encoded amino acids. In addition to the genetically encoded amino acids, the polypeptides described herein may be comprised, either in whole or in part, of naturally occurring and / or synthetic non-encoded amino acids.

[0101] In another aspect, the present disclosure provides polynucleotides encoding the recombinant polypeptides having CYP activity and increased activity and / or yield as described herein. In at least one embodiment, the polynucleotide encoding a recombinant polypeptide having CYP activity comprises an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the polypeptide sequence of SEQ ID NO: 2. In some embodiments, the polynucleotide encodes a recombinant polypeptide comprising an amino acid sequence that has the percent identity described above and has one or more amino acid residue differences as compared to SEQ ID NO: 2 described elsewhere herein.

[0102] In at least one embodiment, the polynucleotide has a sequence encoding a recombinant polypeptide which polynucleotide sequence has one or more neutral codon differences relative to SEQ ID NO: 1, which codon differences do not encode an amino acid difference but result in increased yield of the desired product compound (e.g., UDCA) produced by a recombinant host cell in which the polynucleotide sequence is integrated. In at least one embodiment, the polypeptide is encoded by a polynucleotide sequence having at least 80% identity to SEQ ID NO: 1, and at least one neutral codon difference as compared to SEQ ID NO: 1 at a position encoding an amino acid residue selected from R328, A368, and V476; optionally, wherein the ‐ 41 ‐   neutral codon difference is selected from: R328 (CGT > CGC), A368 (GCT > GCC), and V476 (GTT > GTC).

[0103] It is also contemplated that the polynucleotides encoding the recombinant polypeptides having CYP activity and increased activity and / or yield as described herein, can include a combination of one or more codon differences relative to SEQ ID NO: 1, wherein at least one of the codon differences encodes an amino acid difference as compared to SEQ ID NO: 2 and at least one codon difference is a neutral codon difference that does not encode an amino acid difference as compared to SEQ ID NO: 2 Accordingly, in at least one embodiment, the present disclosure provides a polynucleotide sequence encoding a recombinant polypeptide having CYP activity, wherein the polynucleotide sequence comprises a combination of a codon differences encoding an amino acid difference and a neutral codon difference selected from: R328 (CGT > CGC), A368 (GCT > GCC), and V476 (GTT > GTC).

[0104] In at least one embodiment, the polynucleotide comprises a sequence encoding an exemplary recombinant polypeptide having CYP activity as disclosed in Table 3 and the accompanying Sequence Listing. In at least one embodiment, the polynucleotide comprises a sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 1185, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267, 1269, 1271, and 1275. In at least one embodiment, the polynucleotide comprises a codon degenerate sequence of a sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 1185, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267, 1269, 1271, and 1275.

[0105] The polynucleotide sequences encoding the recombinant polypeptides of the present disclosure may be operatively linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide. Expression constructs containing a heterologous polynucleotide encoding the recombinant polypeptide can be introduced into appropriate host cells to express the corresponding polypeptide. Because of the knowledge of the codons corresponding to the various amino acids, availability of a protein sequence provides a description of all the polynucleotides capable of encoding the subject. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons allows an extremely large number of nucleic acids to be made, all of which encode the improved transaminase enzymes disclosed herein. Thus, having identified a particular amino acid sequence, those skilled in the art could make any number of different nucleic acids by simply modifying the ‐ 42 ‐   sequence of one or more codons in a way which does not change the amino acid sequence of the protein. In this regard, the present disclosure specifically contemplates each and every possible variation of polynucleotides that could be made by selecting combinations based on the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide disclosed herein, including the amino acid sequences presented in Table 3 and the accompanying Sequence Listing.

[0106] The codons can be selected to fit the host cell in which the protein is being produced. For example, preferred codons used in bacteria are used to express the gene in bacteria; preferred codons used in yeast are used for expression in yeast; and preferred codons used in mammals are used for expression in mammalian cells. It is contemplated that all codons need not be replaced to optimize the codon usage of the recombinant polypeptide since the natural sequence will comprise preferred codons and because use of preferred codons may not be required for all amino acid residues. Consequently, codon optimized polynucleotides encoding the recombinant polypeptide may contain preferred codons at about 40%, 50%, 60%, 70%, 80%, or greater than 90% of codon positions of the full-length coding region.

[0107] The present disclosure also provides an expression vector comprising a polynucleotide encoding a recombinant polypeptide having CYP activity and increased thermostability, and one or more expression regulating regions such as a promoter, a terminator, a replication origin, or the like, depending on the type of hosts into which they are to be introduced. The various nucleic acid and control sequences described above 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 recombinant polypeptide at such sites. Alternatively, a polynucleotide sequence of the present disclosure may be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the 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. 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.

[0108] 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 ‐ 43 ‐   contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used. In at least one embodiment, the expression vector further comprises one or more selectable markers, which permit easy selection of transformed cells.

[0109] Use in Recombinant Host Cells

[0110] The engineered genes of the present disclosure that encode recombinant polypeptides with CYP activity (e.g., exemplary polypeptides of Table 3) can be incorporated in recombinant host cells to enable in vivo biosynthesis of compounds cholic acid compounds, such as UDCA and UDCA derivative compounds, that require a cytochrome P450 monooxygenase catalyzed reaction. These recombinant host cells comprise a polynucleotide or expression vector that encodes the recombinant polypeptide with CYP activity, wherein the polynucleotide is operatively linked to one or more control sequences for expression of the polypeptide in the host cell. Host cells for use in expressing recombinant genes encoding the polypeptides with CYP activity of the present disclosure are well known in the art and include but are not limited to, bacterial cells, such as E. coli, or fungal cells, such as Saccharomyces cerevisiae or Pichia pastoris, insect cells, such as Drosophila S2 and Spodoptera Sf9, animal cells, such as CHO, COS, BHK, 293, and plant cells. Appropriate mediums and growth conditions for culturing the recombinant host cells so that they express the polypeptide with CYP activity are well known in the art.

[0111] The recombinant host cells can comprise heterologous nucleic acids encoding not only polypeptides with CYP and CPR activity capable of converting LCA to UDCA (or 3-KCA to 3- KUDCA) but also other enzymes capable of producing other precursor compounds, such as precursors for the substrates, LCA or 3-KCA. As described elsewhere herein, nucleic acid sequences encoding pathway enzymes for producing such precursor compounds are known in the art and can readily be used in accordance with the present disclosure. Typically, the nucleic acid sequence encoding the enzymes which form a part of the pathway, further include one or more additional nucleic acid sequences, for example, a nucleic acid sequence controlling expression of the enzymes which form a part of the biosynthetic pathway, and these one or more additional nucleic acid sequences together with the nucleic acid sequence encoding the polypeptides with CPR and / or CYP activity can be considered a heterologous nucleic acid sequence. A variety of techniques and methodologies are available and well known in the art for introducing heterologous nucleic acid sequences, such as nucleic acid sequences encoding the enzymes (e.g., CPR and CYP), into a host cell so as to attain expression the host cell. Such techniques are well known to the skilled artisan and can be found in, for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, Fourth Ed.

[0112] For example, the introduction of the heterologous nucleic acids can include integration of the nucleic acids into specific loci in the genome of a host cell via CRISPR-Cas9 and other techniques, some of which are demonstrated in the Examples herein. Such techniques are well ‐ 44 ‐   known to the skilled artisan and can, for example, be found in Sambrook and other well-known sources. The number of copies of heterologous genes and their locus of integration in a recombinant host cell’s genome can result in improved biosynthetic production of a desired product, such as UDCA. For example, a heterologous nucleic acid encoding a polypeptide with CYP activity can be integrated in a host cell’s genome in 1, 2, 3, 4, or more copies. Accordingly, it is contemplated that in the recombinant host cells of the present disclosure, the heterologous nucleic acid encoding the recombinant polypeptide having CYP activity can be integrated in the host cell’s genome at one or more loci, including but not limited to the well- known genomic loci in Saccharomyces cerevisiae of X-2, X-4, XI-2, XII-4, NDE1, XII-5, Gal80, and ROQ1, or the loci in Pichia pastoris of AOX1, Int6, Int15, and HIS4.

[0113] One of ordinary skill will recognize that the heterologous nucleic acids encoding the recombinant enzymes with CPR activity and CYP activity, and any other pathway enzymes will further comprise transcriptional promoters capable of controlling expression of the enzymes in the recombinant host cell. Generally, the transcriptional promoters are selected to be compatible with the host cell, so that promoters obtained from bacterial cells are used when a bacterial host cell is selected in accordance herewith, while a fungal promoter is used when a fungal host cell is selected, a plant promoter is used when a plant cell is selected, and so on. Promoters useful in the recombinant host cells of the present disclosure may be constitutive or inducible, provided such promoters are operable in the host cells. Promoters that may be used to control expression in fungal host cells, such as Saccharomyces cerevisiae and Pichia pastoris, are well known in the art and include, but are not limited to inducible promoters, such as a Gal1 promoter or Gal10 promoter, a constitutive promoter, such as an alcohol dehydrogenase (ADH) promoter, a glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, or an S. pombe Nmt, or ADH promoter. Exemplary promoters that may be used to control expression in bacterial cells can include the Escherichia coli promoters lac, tac, trc, trp or the T7 promoter. Exemplary promoters that may be used to control expression in plant cells include, for example, a Cauliflower Mosaic Virus 35S promoter (Odell et al. (1985) Nature 313:810-812), a ubiquitin promoter (U.S. Pat. No.5,510,474; Christensen et al. (1989)), or a rice actin promoter (McElroy et al. (1990) Plant Cell 2:163-171). Exemplary promoters that can be used in mammalian cells include, a viral promoter such as an SV40 promoter or a metallothionine promoter. All of these host cell promoters are well known by and readily available to one of ordinary skill in the art. Further nucleic acid control elements useful for controlling expression in a recombinant host cell can include transcriptional terminators, enhancers, and the like, all of which may be used with the heterologous nucleic acids incorporate in the recombinant host cells of the present disclosure.

[0114] A wide variety of techniques are well known in the art for linking transcriptional promoters and other control elements to heterologous nucleic acid sequences encoding pathway genes for biosynthesis of UDCA or 3-KUDCA. Such techniques are described in e.g., ‐ 45 ‐   Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, Fourth Ed. Accordingly, in at least one embodiment, the heterologous nucleic acid sequences of the present disclosure comprise a promoter capable of controlling expression in a host cell, wherein the promoter is linked to a nucleic acid sequence encoding a recombinant polypeptide having CYP activity of the present disclosure, and as necessary, other enzymes constituting a pathway for production of a UDCA precursor, UDCA, and / or UDCA derivative. This heterologous nucleic acid sequence can be integrated into a recombinant expression vector which ensures good expression in the desired host cell, wherein the expression vector is suitable for expression in a host cell, meaning that the recombinant expression vector comprises the heterologous nucleic acid sequence linked to any genetic elements required to achieve expression in the host cell. Genetic elements that may be included in the expression vector in this regard include a transcriptional termination region, one or more nucleic acid sequences encoding marker genes, one or more origins of replication, and the like. In some embodiments, the expression vector further comprises genetic elements required for the integration of the vector or a portion thereof in the host cell's genome.

[0115] It is also contemplated that in some embodiments an expression vector comprising a heterologous nucleic acid of the present disclosure may further contain a marker gene. Marker genes useful in accordance with the present disclosure include any genes that allow the distinction of transformed cells from non-transformed cells, including all selectable and screenable marker genes. A marker gene may be a resistance marker such as an antibiotic resistance marker against, for example, kanamycin or ampicillin. Screenable markers that may be employed to identify transformants through visual inspection include β-glucuronidase (GUS) (U.S. Pat. Nos.5,268,463 and 5,599,670) and green fluorescent protein (GFP) (Niedz et al., 1995, Plant Cell Rep., 14: 403).

[0116] In at least one embodiment, the present disclosure also provides of a method for producing UDCA or 3-KUDCA, wherein a heterologous nucleic acid encoding a recombinant polypeptide having CYP activity (e.g., an exemplary engineered polypeptide of Table 3) can be introduced into a recombinant host cell. The recombinant host cell can then be used for production of the polypeptide or incorporated in a biocatalytic process that utilized the CYP activity of the recombinant polypeptide expressed by the host cell for the catalytic conversion of a substrate, e.g., the conversion of LCA to UDCA. In at one embodiment, the recombinant host cell can further comprise a pathway of enzymes capable of producing a compound precursor (e.g., LCA) which can act as a substrate for the recombinant polypeptides with CPR and CYP activity. It is contemplated that a recombinant host cell comprising a heterologous nucleic acid encoding a recombinant polypeptide having CYP activity of the present disclosure can provide improved biosynthesis of a desired product compound ( e.g., UDCA or UDCA derivative) in terms of titer, yield, and production rate, due to the improved characteristics of the expressed ‐ 46 ‐   CYP activity in the cell associated with the amino acid and codon differences engineered in the gene.

[0117] Accordingly, in at least one embodiment, the present disclosure provides a method for producing UDCA and / or 3-KUDCA comprising: (a) culturing a recombinant host cell of the present disclosure in a suitable medium comprising LCA and / or 3-KCA; and (b) recovering the produced UDCA and / or 3-KUDCA.

[0118] In at least one embodiment, it is contemplated the recombinant polypeptides with CYP activity of the present disclosure can be incorporated in any biosynthesis method requiring a CYP catalyzed biocatalytic step, whether in vivo or in vitro. Thus, in at least one embodiment, the recombinant polypeptides having CYP activity (e.g., exemplary polypeptides of Table 3) can be used in a method for preparing a compound of structural formula (I)

[0119] In another embodiment, the present disclosure provides a method for preparing a compound of structural formula (I) wherein, R1is substituted C1-C20 alkyl,or an optionally or a is a hydroxyl, or an oxo group. The method comprises contacting a recombinant polypeptide having CYP activity of the present disclosure (e.g., an exemplary polypeptide of Table 3) under suitable reactions conditions with a compound of structural formula (II) 1wherein, R is a an substituted C1-C20 alkyl, or an optionally substituted aryl; R2is hydrogen, or a hydroxyl; and R3is a hydroxyl, or an oxo group. ‐ 47 ‐

[0120] Exemplary conversions of UDCA precursor compounds of structural formula (II) to UDCA and related compounds of structural formula (I) that are catalyzed by the recombinant polypeptides having CYP activity of the present disclosure can include: (1) conversion of LCA to UDCA; (2) the conversion of 3-KCA to 3-KUDCA; (3) the conversion of DCA to UCA. Accordingly, in at least one embodiment of the biosynthesis method for conversion a UDCA precursor compound of structural formula (II) to a UDCA compound of structural formula (I), R2is hydrogen, the compound of structural formula (II) is LCA, and the compound of structure formula (I) is UDCA. In at least one embodiment, R2is hydrogen, the compound of structural formula (II) is 3-KCA and the compound of structure formula (I) is 3-KUDCA. In at least one embodiment, R2is a hydroxyl, the compound of structure formula (II) is DCA, and the compound of structural formula (II) is DCA.

[0121] The present disclosure also contemplates that the methods for biocatalytic conversion of a UDCA precursor compound of structural formula (II) to a UDCA compound of structural formula (I) using an recombinant polypeptide having CYP activity of the present disclosure can comprise additional chemical or biocatalytic steps carried out on the product compound of structural formula (II), including steps of product compound work-up, extraction, isolation, purification, and / or crystallization, each of which can be carried out under a range of conditions.

[0122] Suitable reaction conditions for the biosynthesis of compounds such as UDCA, 3- KUDCA, and UCA, are known in the art and can be used with the recombinant polypeptides having CYP activity of the present disclosure. Additionally, suitable reaction conditions for the exemplary polypeptides of the present disclosure can be determined using routine techniques known in the art for optimizing biocatalytic reactions. It is contemplated that various ranges of suitable reaction conditions with the recombinant polypeptides of the present disclosure, including but not limited to ranges of pH, temperature, buffer, solvent system, substrate loading, polypeptide loading, co-substrate or co-factor loading, atmosphere, and reaction time. Suitable reaction conditions can be readily determined and optimized for particular reactions by routine experimentation that includes, but is not limited to, contacting the recombinant polypeptide and substrate under experimental reaction conditions of concentration, pH, temperature, solvent conditions, and detecting the production of the desired compound of structural formula (I). In at least one embodiment, the suitable reaction conditions comprise a reaction solution of ~pH 7-8, a temperature of 25C to 37C; optionally, the reaction conditions comprise a reaction solution of ~ pH 7 and a temperature of ~30C. In at least one embodiment, the reaction solution is allowed to incubate at a temperature of 25C to 37C for a reaction time of at least 1, 6, 12, 24, or 48 hours, before the amount of reaction product is determined. EXAMPLES

[0123] Various features and embodiments of the disclosure are illustrated in the following representative examples, which are intended to be illustrative, and not limiting. Those skilled in ‐ 48 ‐   the art will readily appreciate that the specific examples are only illustrative of the invention as described more fully in the claims which follow thereafter. Every embodiment and feature described in the application should be understood to be interchangeable and combinable with every embodiment contained within.    Example 1: Genomic Integration of Heterologous CYP / CPR Gene Pairs into Saccharomyces cerevisiae Host Cells

[0124] This example illustrates the preparation of recombinant yeast host cell strains (Saccharomyces cerevisiae), with genomically integrated copies of a pair of heterologous genes encoding a cytochrome P450 (CYP) and a cytochrome reductase (CPR) from Gibberella zeae. This example also demonstrates the ability of these engineered strains to carry out the bioconversion of lithocholic acid (LCA) to ursodeoxycholic acid (UDCA) and / or 3-keto-lithocholic acid (3-KCA) to 3-keto-ursodeoxycholic acid (3-KUDCA).

[0125] A recombinant strain of S. cerevisiae denoted “SAND122,” has been described in International Patent Application publication WO2022115710A1. SAND122 expresses a pair of genes from Gibberella zeae that encode a polypeptide with cytochrome P450 (CYP) activity (SEQ ID NO: 2) and a polypeptide with cytochrome P450 reductase (CPR) activity (SEQ ID NO: 4) and has been shown to carry out the conversion of LCA to UDCA and 3-KCA to 3- KUDCA.

[0126] Material and Methods

[0127] A. S. cerevisiae strain construction

[0128] To improve upon the bioconversion of LCA to UDCA and 3-KCA to 3-KUDCA observed in the strain, SAND122, the wild-type genes encoding the CYP polypeptide from G. zeae of SEQ ID NO: 2 and the CPR polypeptide from G. zeae of SEQ ID NO: 4 were codon-optimized for optimal expression in S. cerevisiae as SEQ ID NO: 1, and SEQ ID NO: 3, respectively. These codon-optimized genes were integrated into the S. cerevisiae genome under the bidirectional pGal1 / 10 promoter system (SEQ ID NO: 59) and ADH1t (SEQ ID NO: 60) and PGKt terminators (SEQ ID NO: 61) to generate single copy strains (SH010 and SH013) as follows.

[0129] Nucleic acids with the codon-optimized CYP gene of SEQ ID NO: 1, and the codon optimized CPR gene of SEQ ID NO: 3 were synthesized as fragments (Twist Bioscience, Inc., South San Francisco, California), which were further PCR amplified to incorporate ~20-30 bp overhang sequences homologous to the bidirectional pGal1 / 10 promoter and the respective terminator sequences on the 5’ or 3’ ends of the respective gene sequences. Locus specific PCR amplicons were also amplified with ~20-30 bp homologous overhangs to the terminator regions to enable efficient integration into the S. cerevisiae X-4 locus. The PCR primer sequences used in the following S. cerevisiae strain building examples by their Primer#, are listed in Table 4 below. ‐ 49 ‐

[0130] TABLE 4 Primer SEQ # Primer Sequence ID NO: P1ACTGCAAAGGCGTGCCCAAA62

[0131] With reference to the primer numbers listed in Table 4 and as illustrated in Scheme A of FIG.1, Fragment A encompassing the X-4 site upstream homology region, ADH1t terminator, and CPR was amplified using the forward primer P1 and the reverse primer P2 as illustrated in ‐ 50 ‐   Scheme A of FIG.1. Fragment B encompassing the bidirectional pGal1 / 10 promoter was amplified using the forward primer P3 and the reverse primer P4. Fragment C containing the CYP gene was amplified using the forward primer P5 and the reverse primer P6. Fragment D containing the PGK1t terminator was amplified using the forward primer P7 and the reverse primer P8. Fragment E containing the X-4 downstream homology region was amplified using the forward primer P9 and the reverse primer P10. The five fragments A-E were assembled by NEBuilder® HiFi Assembly and amplified as a single linear DONOR fragment using the following rescue forward primer P11 and the rescue reverse primer P12. The final assembled 4680 bp linear DONOR DNA was gel extracted and integrated as a knock-in using CRISPR- Cas9 into the X-4 locus of the yeast strain CENPK2-1D. This SH010 strain was used as the parent strain to introduce a gal80 knockout to enable induction of the bidirectional promoter pGal1 / 10 in the absence of glucose. The Gal80 knockout DONOR DNA was amplified using the forward rescue primer P13 and the reverse rescue primer P14 and integrated as a knock-in using CRISPR-Cas9 to disrupt the native gal80 gene in SH010 resulting in the ∆Gal80, single copy CYP-CPR strain SH013.

[0132] PCR primers summarized in Table 4 also were used to amplify the above-mentioned parts as follows and illustrated by Scheme B of FIG.1. Recombinant host cell strains with double (SHP021), triple (SH020), and quadruple (SHP025) copies of the CYP / CPR cassette integrated in the genome were also built. To generate the multi-copy strains, upstream and downstream homology regions for the XI-2, XII-4, and X-2 loci were assembled to the 5’ and 3’ ends of the central DNA fragment (Fragment B) encompassing the CPR / CYP genes under the bidirectional pGAL1 / 10 promoter and respective terminators (found in strain SH013). Fragment B was amplified using the forward primer P15 and the reverse primer P16. For the XI-2 locus, the 5’ upstream homology arm (Fragment A) was amplified using the forward primer P17 and the reverse primer P18 and the 3’ downstream homology arm (Fragment C) was amplified using the forward primer P19 and the reverse primer P20. For the XII-4 locus, 5’ upstream homology arm (Fragment A) was amplified using the forward primer P21 and the reverse primer P22 and the 3’ downstream homology arm (Fragment C) was amplified using the forward primer P23 and the reverse primer P24. For the X-2 locus, 5’ upstream homology arm (Fragment A) was amplified using the forward primer P25 and the reverse primer P26 and the 3’ downstream homology arm (Fragment C) was amplified using the forward primer P39 (SEQ ID NO: 100) and the reverse primer P40 (SEQ ID NO: 101). The three fragments A, B, and C were assembled by NEBuilder® HiFi Assembly and amplified as a single linear DONOR DNA using the following site-specific primers: forward primer P27 and reverse primer P28 for the XI- 2 DONOR, forward primer P29 and reverse primer P30 for the XII-4 DONOR, and forward primer P31 and reverse primer P32 for the X-2 DONOR. The XI-2 DONOR DNA was transformed and integrated into XI-2 locus as a knock-in using CRISPR-Cas9 into the SH013 ‐ 51 ‐   (single copy) strain to generate the double copy strain SHP021. The XII-4 DONOR was transformed and integrated into XII-4 locus as a knock-in using CRISPR-Cas9 into the SHP021 (double copy) strain to generate the triple copy strain SH020. The X-2 DONOR was transformed and integrated into X-2 locus as a knock-in using CRISPR-Cas9 into the SH020 (triple copy) strain to generate the quadruple copy strain SHP025.

[0133] The lineage for the four above-described strains (SH013, SH021, SH020, and SHP025) constructed in Saccharomyces cerevisiae is summarized in FIG.2.

[0134] B. Screening of strains for 3-KCA to 3-KUDCA conversion

[0135] A HTP screening assay of the recombinant host cell strains for the bioconversion of 3- KCA to 3-KUDCA was developed. 3-KCA to 3-KUDCA conversion over LCA to UDCA conversion was used to determine activity in HTP as 3-KCA was found to have better solubility than LCA in the bioconversion reaction buffer. The HTP screening assay was carried out as follows: Individual colonies of the recombinant S. cerevisiae strains were picked into 500 mL baffled conical flasks containing 2 x YPD media (100 mL working volume). The flasks were incubated at 30oC for 48 h with shaking at 250 rpm at 85 % humidity. After this time, a 1% final concentration solution of galactose (2.5 mL of a 40 % stock solution) was added and the flask was further incubated for 4 hours to induce protein production. After this induction period, the biomass was harvested via centrifugation and the supernatant removed. A portion of the resulting biomass (approximately 2 g) was resuspended in 10 mL of bioconversion buffer (295 mg / L 3-KCA, 0.1 M phosphate buffer, pH 9) and the flask was incubated at 30oC for 24 h with shaking at 250 rpm at 85 % humidity. After this time, an aliquot of the reaction mixture (1 mL) was extracted with a 1:1 ratio of MeOH (1 mL) with shaking at 30oC, 250 rpm and 85 % humidity for 30 minutes. The biomass was then removed via centrifugation and the supernatant diluted further with MeOH (1000 x final dilution) before analysis using an Agilent 6470 Triple Quadrupole LC / MS.

[0136] LC / MS sample preparation: The bioconversion reaction was extracted and diluted with MeOH for sample preparation as described above. The prepared samples were loaded onto an Agilent 6470 Triple Quadrupole LC / MS and the compounds of interest were detected using MS / MS in SIM mode. The compounds were quantified relative to calibration curves which were prepared using serial dilutions of stock solutions of 3-KCA and 3-KUDCA.

[0137] LC / MS instrumentation and parameters: LC / MS system: Agilent 6470 Triple Quadrupole LC / MS; Column: Agilent EclipsePlusC18 column 1.8 um, 3.0 x 50 mm; Mobile phase: Solvent A: H2O with 0.1% formic acid; Solvent B: Acetonitrile with 0.1% formic acid; Gradient: 0 - 0.2 min isocratic 40% B, 0.2 - 2.2 min gradient to 85% B, 2.2 - 2.8 min isocratic 85% B; Mode: MS / MS in SIM mode (monitoring at 389.3 for 3-KUDCA and 373.2 for 3-KCA); Fragmentor: 90 volts.

[0138] Results ‐ 52 ‐

[0139] Results of screening assays for the four S. cerevisiae strain builds are summarized in Table 5 below.

[0140] TABLE 5 3-KCA to 3-KUDCA S. cerevisiae Strain % Conversion @295mg / LHost Cells by Site Saturation Mutagenesis (SSM)

[0141] This example illustrates the preparation of site saturation mutagenesis (SSM) libraries of engineered polypeptides derived from the parent CYP polypeptide of SEQ ID NO: 2 in the SH013 strain from Example 1 and screening these variant strains for improved activity in the bioconversion of 3-KCA to 3-KUDCA relative to the bioconversion of the parent strain SH013.

[0142] Material and Methods

[0143] A. Site Saturation Mutagenesis library construction

[0144] The heterologous nucleic acid sequence from the single copy strain SH013 build of Example 1, which includes the codon-optimized polynucleotide sequence of SEQ ID NO:1, which encodes the CYP polypeptide of SEQ ID NO: 2, expressed under the pGal1 / 10 promoter system and PGK1t terminator, was used to design SSM oligonucleotide to generate SSM libraries at positions spanning the entire polypeptide sequence. To generate an appropriate screening strain to evaluate these libraries, the URA3 marker was integrated into the X-4 site (Easy-Clone 2.0) of a yeast strain under the pGal1 promoter within the pGal1 / 10 promoter system (SEQ ID NO: 59) and PGKt1 terminator (SEQ ID NO: 61), along with the codon- optimized polynucleotide sequence of SEQ ID NO: 3, which encodes the CPR polypeptide of SEQ ID NO: 4, under the pGal10 promoter within the pGal1 / 10 promoter system (SEQ ID NO: 59) and ADH1t terminator (SEQ ID NO: 60). The resulting strain SHEVP003 was used as the negative control and screening host for CYP SSM library integration. Libraries were plated on selective media containing 5’-fluoroorotic acid, which selects for URA3 negative cells, indicating successful integration of DONOR DNA into the SHEVP003 screening strain. SH013 strain was ‐ 53 ‐   used as a control strain during screening of the SSM library strains for fold-improvement calculations with respect to conversion of 3-KCA to 3-KUDCA as described below.

[0145] Genomic DNA from the SH013 strain was used as the template to generate two PCR products: (1) a first PCR product (Fragment A), which does not harbor any degenerate codons, and (2) a second PCR product (Fragment B), which has sequence overlap with the Fragment A, and is amplified harboring one NNK degenerate codon only. Primers spanning the full 513 codons of the CYP polypeptide were designed according to standard site-saturation mutagenesis protocols and used for amplification of Fragments A and B and overlap extension.

[0146] Fragment A was amplified using a single forward primer P34 (SEQ ID NO: 95) and a series of 513 reverse primers designed according to the location of the desired mutagenesis site in CYP. The 513 reverse primers for Fragment A listed in Table 6A (below) and are provided in the accompanying Sequence Listing as SEQ ID NOs: 102-614.

[0147] TABLE 6A: Reverse primers for SSM library Fragment A amplification SEQ ID Primer Name Primer Sequence NO:‐ 54 ‐ Codon364_C5AGGGCGAAGTCTCTGTGACTCTTTTATGAC130Codon365_C6AACAGGGCGAAGTCTCTGTGACTCTTTTAT131‐ 55 ‐ Codon138_C4GTTAACGAGCGGCACCAGATCGAACGGGGA176Codon139_C5CTTGTTAACGAGCGGCACCAGATCGAACGG177‐ 56 ‐ Codon379_G2GGTAACATCAGCCAATGCCATTCTCCTGAA222Codon387_G3TTTAATTACGTCGCCATTGGGCAAGGTAAC223‐ 57 ‐ Codon124_B12GAATCCAGGAATATAACCGTGAGTATCGTC268Codon125_C1CTCGAATCCAGGAATATAACCGTGAGTATC269‐ 58 ‐ Codon373_F10CATTCTCCTGAAAGCACCTAATAAAACAGG314Codon375_F11CAATGCCATTCTCCTGAAAGCACCTAATAA315‐ 59 ‐ Codon121_B8AATATAACCGTGAGTATCGTCTCTTGCGAC360Codon128_B9ACCGATTGGCTCGAATCCAGGAATATAACC361‐ 60 ‐ Codon325_F6AAGGAGTTCTGGTTGCAAAGCTATATTAAA406Codon340_F7ACCTTCCGTGGATAAAACCGTAACTATCTC407‐ 61 ‐ Codon50_B4AAAAGGTTTCTTAGGGTTGATATAAGGATA452Codon52_B5CAGCTCAAAAGGTTTCTTAGGGTTGATATA453‐ 62 ‐ Codon312_F2AGACAGTAAATCAGTTGTTGTGTGAATCGC498Codon317_F3ATTAAACATTGTTTCAGACAGTAAATCAGT499‐ 63 ‐ Codon70_A12AGTCAAAATGTCTCGTGCGTTCTCGATAAA544Codon75_B1AAGTGAGCGACCCTTAGTCAAAATGTCTCG545‐ 64 ‐   Codon369_E10AGCACCTAATAAAACAGGGCGAAGTCTCTG590Codon385_E11TACGTCGCCATTGGGCAAGGTAACATCAGC591g p g p series of 370 forward primers that included a single NNK degenerate codon spanning across successive positions of the CYP encoding gene of SEQ ID NO: 1. The 370 forward primers for Fragment B are listed in Table 6B (below) and are provided in the accompanying Sequence Listing as SEQ ID NOs: 615-984.

[0149] TABLE 6B: Forward primers for SSM library Fragment B amplification SEQ ID :‐ 65 ‐ Codon47_A11GGCAAGCAATATCCTTATATCAACCCTAAGNNKCCTTTTGAGCTG625Codon66_A12GTTGTCCAGGATTTTATCGAGAACGCACGANNKATTTTGACTAAG6267‐ 66 ‐ Codon314_E9CACACAACAACTGATTTACTGTCTGAAACANNKTTTAATATAGCT671Codon323_E10ACAATGTTTAATATAGCTTTGCAACCAGAANNKCTTGGTCCACTA672‐ 67 ‐ Codon31_A7ACTTTATTTAGCTTTTTCATACTAAAGTATNNKCTTCTCGGAAAC717Codon32_A8TTATTTAGCTTTTTCATACTAAAGTATAGTNNKCTCGGAAACGGG718‐ 68 ‐ Codon288_E5ATAGAATGGTTTGAAAATGAATACGAGGGCNNKTCTGATCCCGCC763Codon294_E6GAATACGAGGGCAAATCTGATCCCGCCACTNNKCAAATTAAACTA764‐ 69 ‐ Codon14_A3GATCTAGACCTAGTATTAGGAAAAAGTCAANNKGCATTATTTTGT809Codon16_A4GACCTAGTATTAGGAAAAAGTCAATACGCANNKTTTTGTGGCATA8101‐ 70 ‐ Codon243_E1TTTTTGCCTTCCTGTCAAGGGGTTAGGAGANNKTTGCAGGAAGCG855Codon247_E2TGTCAAGGGGTTAGGAGAAAATTGCAGGAANNKCGTGATTTATTG8567‐ 71 ‐ Codon509_H11AGGAGAGCAGCCGAAATCGATATAGACACTNNKGGTAGTGATGAA901Codon511_H12GCAGCCGAAATCGATATAGACACTATTGGTNNKGATGAATAAATT902‐ 72 ‐   Codon276_D9ATCGCTGAAGGTAGACCATCACCATTCGACNNKTCAATAGAATGG947Codon280_D10AGACCATCACCATTCGACGATTCAATAGAANNKTTTGAAAATGAA948

[0150] The two fragments A and B were assembled by overlap extension PCR using forward primer P35 (SEQ ID NO: 96) and reverse primer of P36 (SEQ ID NO: 97). The assembled OE- PCR products were then pooled together, and gel purified to provide a saturation mutagenesis library of linear donor DNA. The pooled saturation mutagenesis library linear donor DNA was integrated as a knock-in using CRISPR-Cas9 into the X-4 locus where URA3 was integrated in SHEVP003 which already had the CPR gene integrated. ‐ 73 ‐

[0151] B. Screening of SSM library

[0152] Screening of the recombinant host cells for bioconversion of 3-KCA to 3-KUDCA was carried out according to a protocol adapted from that described in Example 1. Individual colonies of recombinant S. cerevisiae strains were picked into 96-well plates containing 2 x YPD media (300 ^L per well) using a QPixTM420 colony picking system. The plates were incubated at 30oC for 24 h with shaking at 250 rpm at 85 % humidity. After this time, the strains were sub-cultured (40 ^L inoculation volume per strain) into a second 96-well plate containing 2 x YPD media (1.2 mL per well) using an Agilent Bravo automated liquid handling platform. The plates were incubated at 30oC for 48 h with shaking at 250 rpm at 85 % humidity. After this time, a solution of galactose (40 % stock solution) was added using the Bravo (1 % final concentration) and the plates were further incubated for 4 hours to induce protein production. After this time, the biomass was harvested via centrifugation and the supernatant removed. The resulting biomass was resuspended in 300 ^L of bioconversion buffer (295 mg / L 3-KCA, 0.1 M phosphate buffer, pH 9) using the Agilent Bravo automated liquid handling platform and the plates were incubated at 30oC for 24 h with shaking at 250 rpm at 85 % humidity. After this time, MeOH (300 ^L) was added using the Bravo and the plates were incubated at 30oC with shaking at 250 rpm at 85 % humidity for a further 30 minutes. The biomass was then separated by centrifugation and the resulting supernatant diluted with MeOH (1000 x total dilution) using the Agilent Bravo automated liquid handling platform before analysis using an Agilent 6470 Triple Quadrupole LC / MS. Sample preparation and analytical methods were identical to those described in Example 1.

[0153] Results

[0154] Data from screening the SSM libraries in terms of fold-improvement in production of 3- KUDCA from 3-KCA relative to the control strain, SH013 are summarized in Table 7 (below).

[0155] TABLE 7 Name FIOC n       29 30 G325E 11 0.40Host Cells by Combinatorial Mutagenesis

[0156] This example illustrates the preparation of further combinatorial libraries of CYP genes based on the parent strain SHS036 which carries a gene encoding the S31V mutant of CYP (SEQ ID NO: 6) identified through SSM in Example 2 for improved bioconversion of 3-KCA to 3- KUDCA.

[0157] Material and Methods

[0158] A. Combinatorial library construction

[0159] The polynucleotide sequence of SEQ ID NO: 5 encoding the S31V mutant CYP polypeptide (SEQ ID NO: 6) from SHS036 expressed under the pGal1 promoter within the pGal1 / 10 promoter system (SEQ ID NO: 59) and PGK1t terminator (SEQ ID NO: 61), was used to design oligos to generate combinatorial libraries to randomly incorporate additional identified beneficial amino acid mutations from Example 2 into the S31V CYP backbone sequence. The same screening strain was used to integrate the resulting libraries as in Example 2 (SHEVP003), however the SHS036 strain was used as the parent control strain in order to determine fold-improvement in conversion of 3-KCA to 3-KUDCA as described below.

[0160] A semi-synthetic approach was used to construct the first set of combinatorial libraries. Genomic DNA from the SHS036 strain was used as the template to generate a full-length PCR product using primer pair of P34 (SEQ ID NO: 95) and P37 (SEQ ID NO: 98) while incorporating uracil using a dNTP mix comprising of the following deoxyribonucleotides: dATP, dGTP, dCTP, dTTP, dUTP. The resulting PCR product was gel purified and digested with Uracil-DNA Glycosylase and Endonuclease IV at 37 C for 2 hours, followed by enzyme denaturation at 94 C for two minutes, to generate a pool of fragments in the range of 50-100 bases. These fragments were further combined with differing ratios of pools of the synthesized oligonucleotide primers (each oligo up to 55 bases in length and encoding one or more amino acid change) in several individual assembly PCR reactions using forward primer P5 (SEQ ID NO: 66) and reverse primer P6 (SEQ ID NO: 67) to reassemble the full-length PCR product as Fragment B and incorporate mutagenic amino acid changes within each pool randomly. The synthesized oligonucleotide primers SEQ ID NOs: 985-1001 used in the pools and their encoded mutations are listed in Table 8 below and the accompanying Sequence Listing.

[0161] TABLE 8 Mutation SEQ ID N L th Pi S NO‐ 75 ‐   CYP_CMB1_V56G 49AACCTTTTGAGCTGTCGAATCAACGAGGAGTC987 p3 CAGGATTTTATCGAGAA

[0006] or e cent ntegraton o t e combnatora mutant C varants, C amp cons wth homologous 5’ and 3’ regions to the CYP were generated (Fragment A and Fragment C respectively). Fragment A was amplified using the forward primer P37 (SEQ ID NO: 98) and reverse primer P4 (SEQ ID NO: 65) and Fragment C was amplified using the forward primer P7 (SEQ ID NO: 68) and the reverse primer P38 (SEQ ID NO: 99). Fragments A, B, and C were assembled by NEBuilder® HiFi Assembly and amplified as a single linear DONOR using the following forward rescue primer P35 (SEQ ID NO: 96) and reverse primer P36 (SEQ ID NO: 97). The assembled PCR products were then pooled together, and gel purified to provide a combinatorial library of linear donor DNA.

[0163] The pooled combinatorial libraries of linear donor DNA were integrated as a knock-in using CRISPR-Cas9 into the X-4 locus where URA3 was integrated in SHEVP003 which already had the CPR gene integrated as in Examples 1 and 2. ‐ 76 ‐

[0164] B. Screening of Combi libraries

[0165] Screening of the recombinant host cells for bioconversion of 3-KCA to 3-KUDCA was carried out using the same protocol described in Example 2 except the 3-KCA loading was increased from 295 mg / L to 590 mg / L.

[0166] Results

[0167] Screening of the combinatorial libraries for fold-improvement in production of 3-KUDCA from 3-KCA relative to the control strain, SHS036 which expresses the S31V CYP polypeptide of SEQ ID NO: 6 (also referred to herein as “CYP100”) and the CPR polypeptide of SEQ ID NO: 4 (also referred to herein as “CPR001”), are summarized in Table 9 (below).

[0168] TABLE 9 3-KCA to 3- FIOC NT AA KUDCA 3-KCA to 3- nExample 4: Genomic Integration of Heterologous CYP / CPR Gene Pairs into Pichia pastoris Host Cells

[0169] This example illustrates the preparation of recombinant yeast host cell strains (Pichia pastoris) with genomically integrated copies of a pair of heterologous genes encoding a ‐ 77 ‐   cytochrome P450 (CYP) and cytochrome reductase (CPR) enzymes from Gibberella zeae and demonstration of the ability of these strains to carry out the bioconversion of Lithocholic acid (LCA) to Ursodeoxycholic acid (UDCA) and / or 3-Keto-lithocholic acid (3-KCA ) to 3-Keto- ursodeoxycholic acid (3-KUDCA).

[0170] A recombinant strain of P. pastoris, denoted as “SAND121” has been described previously in WO2022115710A1. SAND121 expresses a pair of genes encoding a cytochrome P450 (CYP) and a reductase (CPR) from Gibberella zeae and has been shown to carry out the conversion of LCA to UDCA and 3-KCA to 3-KUDCA.

[0171] Material and Methods

[0172] A. P. pastoris strain building

[0173] To improve upon the bioconversion of LCA to UDCA and 3-KCA to 3-KUDCA observed in the SAND121 strain, yeast codon-optimized polynucleotide sequences of SEQ ID NO: 1 and 3 encoding the CYP polypeptide of SEQ ID NO: 2 and the CPR polypeptide of SEQ ID NO: 4, respectively, were integrated into the P. pastoris genome under the bidirectional pCAT1:pFDH1 promoter system (SEQ ID NO: 1002) and tDAS1 and tDAS2 terminators SEQ ID NO: 1003 and SEQ ID NO: 1004, respectively, to generate a single copy strain (SHP026) as follows. A single plasmid (wbplasmid147; SEQ ID NO: 1059) containing a zeocin resistance marker, a pUC origin of replication, a HIS4 homologous sequence (SEQ ID NO: 1005), and a cassette comprising of tDAS1:CPR001:pCAT1:pFDH1:CYP001:tDAS2 was assembled. Sequences were amplified using PCR using the following primers: wboligos5777, wboligos5792, wboligos5793, wboligos5796, wboligos5797, wboligos5798, wboligos5800, wboligos5801, wboligos5802, wboligos5803, wboligos5793, wboligos5794, wboligos5795, and wboligos5776.

[0174] The sequences of the PCR primers described in the following P. pastoris strain building examples by their Primer Name are listed in Table 10 below and the accompanying Sequence Listing.

[0175] TABLE 10 SEQ ID : 6 7 8 9 0 1‐ 78 ‐ wboligos5796TTCGAAAAGTCTCAAATCCATACTTTTTAAACGGGAAGTCTTTACAGT1012 TTTAGTTAGGAG 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4‐ 79 ‐   wboligos5995ACCATGTTTCGTTGGAAGGGAGATGCTGAAAGAGATTTGGCTATCCTT1035 AATTGCTCCTTC 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0

[0176] PCR amplification was followed by agarose-gel purification and assembly using NEB’s HiFi DNA Assembly Master Mix (catalog no. E2621X) according to manufacturer’s instructions. Since the PCR amplicons had overlapping ends, this allowed for whole plasmid assembly. Three microliters of the HiFi assembly were used to transform E. coli competent cells and plated on low-salt LB media containing zeocin. Plasmids were extracted from cultures inoculated by colonies using Thermo-Fisher GeneJet Plasmid Miniprep Kit (catalog no. K0503) and sequenced to confirm correct plasmid assembly. One sequence-confirmed plasmid was digested with BamHI and further purified using Zymo Research’s DNA Clean and Concentrator Kit (catalog no. D4014). The restriction enzyme cut site found in the HIS4 homologous sequence, linearized the plasmid, allowing for homologous integration at the HIS4 locus in Pichia competent cells, which were prepared from the wild-type BG-10 strain using Thermo- ‐ 80 ‐   Fisher Pichia EasyComp Transformation Kit (catalog no. K173001) according to manufacturer’s instructions.5 microliters of linearized plasmid were used to transform 50 μL of BG-10 competent cells and plated on YPDS media plates containing zeocin. After 3 days of growth, individual colonies were confirmed for integration of the linearized plasmid using PCR and genomic DNA isolated from individual colonies.

[0177] A recombinant Pichia host cell strain (SHP029) with two copies of a gene encoding CYP (SEQ ID NO: 2) and one copy of a gene encoding CPR (SEQ ID NO: 4) was also constructed as follows. A single plasmid (wbplasmid171; SEQ ID NO: 1061) containing an ampicillin resistance marker, a G418 resistance marker, a pUC origin of replication, promoter pAOX1 (SEQ ID NO: 1051), CYP (SEQ ID NO: 2), and terminator tDAS2 (SEQ ID NO: 1004) was assembled using the method described above. Sequences were amplified using PCR with the following primers: wboligos5979, wboligos6014, wboligos6013, wboligos6015, wboligos6016, wboligos5921, wboligos5922, wboligos5802, wboligos5803, and wboligos5978. Following plasmid recovery and sequence confirmation as described above, a single plasmid was digested with SacI and further purified using Zymo Research’s DNA Clean and Concentrator Kit (catalog no. D4014). The restriction enzyme cut site, found in pAOX1, linearized the plasmid, allowing for homologous integration at the AOX1 locus. Five microliters of linearized plasmid were used to transform 50 μL of SHP026 competent cells and plated on YPD media plates containing G418. After 3 days of growth, individual colonies were confirmed for integration of the linearized plasmid using PCR and genomic DNA from each individual colony.

[0178] A recombinant Pichia host cell strain (SHP030) with one copy of a gene encoding the S31V mutant of CYP (SEQ ID NO: 6) and one copy of the gene encoding CPR (SEQ ID NO: 4) was also constructed as follows. A single plasmid (wbplasmid160; SEQ ID NO: 1060) containing a zeocin resistance marker, a pUC origin of replication, a HIS4 homologous sequence (SEQ ID NO: 1005), and a cassette comprising tDAS1:CPR:pCAT1:pFDH1:CYP100:tDAS2 was assembled as described above. As wbplasmid147 shares the same sequence as wbplasmid160 except for the CYP CDS, inverse PCR was used to amplify the vector excluding the CYP CDS using primers wboligos5800 and wboligos5803. CYP (SEQ ID NO: 2) was amplified from SHS036 gDNA using primers wboligos5801 and wboligos5802. Following plasmid recovery and sequence confirmation, a single plasmid was digested with BamHI and further purified using Zymo Research’s DNA Clean and Concentrator Kit (catalog no. D4014). The restriction enzyme cut site, found in the HIS4 homologous sequence, linearized the plasmid, allowing for homologous integration at the HIS4 locus. Five microliters of linearized plasmid were used to transform 50 μL of BG-10 competent cells and plated on YPDS media plates containing zeocin. After 3 days of growth, individual colonies were confirmed for integration of the linearized plasmid using PCR and genomic DNA from each individual colony. ‐ 81 ‐

[0179] A recombinant Pichia host cell strain (SHP033) with two copies of the gene encoding CYP (SEQ ID NO: 2) and one copy of a gene encoding CPR (SEQ ID NO: 4) was also constructed as follows. A single plasmid (wbplasmid172; SEQ ID NO: 1062) containing an ampicillin resistance marker, a G418 resistance marker, a pUC origin of replication, promoter pAOX1 (SEQ ID NO:1051), gene encoding CYP100 (SEQ ID NO: 6), and terminator tDAS2 (SEQ ID NO: 1004) was assembled with the method described above. As wbplasmid171 shares the same sequence as wbplasmid172 except for the CYP CDS, inverse PCR was used to amplify the vector excluding the CYP CDS using primers wboligos5921 and wboligos5803. CYP was amplified from SHS036 gDNA using primers wboligos5922 and wboligos5802. Following plasmid recovery and sequence confirmation as described above, a single plasmid was digested with SacI and further purified using Zymo Research’s DNA Clean and Concentrator Kit (catalog no. D4014). The restriction enzyme cut site, found in pAOX1, linearized the plasmid, allowing for homologous integration at the AOX1 locus. Five microliters of linearized plasmid were used to transform 50 μL of SHP030 competent cells and plated on YPD media plates containing G418. After 3 days of growth, individual colonies were confirmed for integration of the linearized plasmid using PCR and genomic DNA from each individual colony.

[0180] A recombinant Pichia host cell strain (SHP034) with three copies of a gene encoding the S31V CYP (SEQ ID NO: 6) and two copies of the gene encoding CPR (SEQ ID NO: 4) was also constructed as follows. A single plasmid (wbplasmid173; SEQ ID NO:1063) containing a hygromycin resistance marker, an ampicillin resistance marker, a pUC origin of replication, an Int6 homologous sequence (SEQ ID NO: 1052), and a cassette comprising terminator tPMP20 (SEQ ID NO: 1053), CPR (SEQ ID NO: 4), bidirectional promoter pCAT1:FDH1 (SEQ ID NO: 1054), CYP100 (SEQ ID NO: 6), and terminator tFLD1 (SEQ ID NO: 1055), was assembled as described above. Sequences were amplified using PCR using the following primers: wboligos5983, wboligos5984, wboligos5985, wboligos5986, wboligos5987, wboligos5988, wboligos5989, wboligos5990, wboligos5991, and wboligos5992. Following plasmid recovery and sequence confirmation as described above, a single plasmid was digested with BsgI and further purified using Zymo Research’s DNA Clean and Concentrator Kit (catalog no. D4014). The restriction enzyme cut site, found in the Int6 homologous sequence, linearized the plasmid, allowing for homologous integration at its corresponding locus (an intergenic region). Five microliters of linearized plasmid were used to transform 50 μL of SHP033 competent cells and plated on YPD media plates containing hygromycin. After 3 days of growth, individual colonies were confirmed for integration of the linearized plasmid using PCR and genomic DNA from each individual colony.

[0181] A recombinant Pichia host cell strain (SHP035) with four copies of the gene encoding S31V CYP (SEQ ID NO: 6) and three copies of the gene encoding CPR (SEQ ID NO: 4) was also constructed as follows. A single plasmid (wbplasmid174; SEQ ID NO:1064) containing a ‐ 82 ‐   nourseothricin resistance marker, an ampicillin resistance marker, a pUC origin of replication, an Int15 homologous sequence (SEQ ID NO: 1056), and a cassette comprising of terminator tFBA2 (SEQ ID NO: 1057), gene encoding CPR (SEQ ID NO:4), bidirectional promoter pCAT1:FDH1 (SEQ ID: 1053), gene encoding CYP100 (SEQ ID NO: 6), and terminator tADH2 (SEQ ID NO: 1058), was assembled as described above. Sequences were amplified using PCR using the following primers: wboligos5993, wboligos5994, wboligos5995, wboligos5996, wboligos5997, wboligos5998, wboligos5999, wboligos6000, wboligos6001, and wboligos6002. Following plasmid recovery and sequence confirmation as described above, a single plasmid was digested with SalI and further purified using Zymo Research’s DNA Clean and Concentrator Kit (catalog no. D4014). The restriction enzyme cut site, found in the Int15 homologous sequence, linearized the plasmid, allowing for homologous integration at its corresponding locus (an intergenic region). Five microliters of linearized wbplasmid173 and 5 microliters of linearized wbplasmid174 were used to co-transform 50 μL of SHP033 competent cells and plated on YPD media plates containing hygromycin and nourseothricin. After 3 days of growth, individual colonies were confirmed for integration of the linearized plasmids using PCR and genomic DNA from each individual colony.

[0182] A recombinant Pichia host cell strain (SHP038) with one copy of a polynucleotide of SEQ ID NO: 31 encoding the CYP105 mutant (S31V, Q54K, A143G, T307A, G325E) of SEQ ID NO: 32 identified in Example 3 and one copy of the gene of SEQ ID NO: 3 encoding CPR (SEQ ID NO: 4) was also constructed as follows. A single plasmid (wbplasmid193; SEQ ID NO: 1065) containing a zeocin resistance marker, a pUC origin of replication, a HIS4 homologous sequence (SEQ ID NO: 1005), and a cassette comprising of tDAS1:CPR001:pCAT1:pFDH1:CYP105:tDAS2 was assembled as described above. As wbplasmid147 shares the same sequence as wbplasmid193 except for the CYP CDS, inverse PCR was used to amplify the vector minus the CYP CDS using primers wboligos5800 and wboligos5803. CYP105 (SEQ ID NO: 32) was amplified from SHS042 gDNA using primers wboligos5801 and wboligos5802. Following plasmid recovery and sequence confirmation as described above, a single plasmid was digested with BbvcI and further purified using Zymo Research’s DNA Clean and Concentrator Kit (catalog no. D4014). The restriction enzyme cut site, found in the HIS4 homologous sequence, linearized the plasmid, allowing for homologous integration at the HIS4 locus. Five microliters of linearized plasmid were used to transform 50 μL of BG-10 competent cells and plated on YPDS media plates containing zeocin. After 3 days of growth, individual colonies were confirmed for integration of the linearized plasmid using PCR and genomic DNA from each individual colony.

[0183] A recombinant Pichia host cell strain (SHP052) with one copy of a gene encoding CYP105 mutant (S31V, Q54K, A143G, T307A, G325E) of SEQ ID NO: 32 identified in Example 3 was also constructed as follows. A single plasmid (wbplasmid202; SEQ ID NO:1066) containing a geneticin resistance marker, an ampicillin resistance marker, a pUC origin of ‐ 83 ‐   replication, and a cassette comprising of pAOX1:CYP105:tDAS2 was assembled as described above. As wbplasmid202 shares the same sequence as wbplasmid160 except for the CYP100 CDS, inverse PCR was used to amplify the vector minus the CYP100 CDS using primers wboligos5803 and wboligos5921. CYP105 (SEQ ID NO: 32) was amplified from SHS042 gDNA using primers wboligos5922 and wboligos5802. Following plasmid recovery and sequence confirmation as described above, a single plasmid was digested with SacI and further purified using Zymo Research’s DNA Clean and Concentrator Kit (catalog no. D4014). The restriction enzyme cut site, found in the Int6 homologous sequence, linearized the plasmid, allowing for homologous integration at the Int6 locus (intergenic region). Five microliters of linearized plasmid were used to transform 50 μL of BG-10 competent cells and plated on YPDS media plates containing geneticin. After 3 days of growth, individual colonies were confirmed for integration of the linearized plasmid using PCR and genomic DNA from each individual colony.

[0184] A recombinant Pichia host cell strain (SHP053) with two copies of the gene encoding CYP105 (SEQ ID NO: 32) identified in Example 3 and one copy of the gene encoding CPR (SEQ ID NO: 4) was also constructed as follows. Plasmid wbplasmid202 was assembled, digested, and purified as described above. Five microliters of linearized plasmid were used to transform 50 μL of SHP038 competent cells and plated on YPDS media plates containing geneticin. After 3 days of growth, individual colonies were confirmed for integration of the linearized plasmid using PCR and genomic DNA from each individual colony.

[0185] The strain build lineage for all nine of the above-described P. pastoris strains is summarized in the chart of FIG.3.

[0186] B. Screening of strains for 3-KCA to 3-KUDCA conversion

[0187] Screening of the recombinant host cells for bioconversion of 3-KCA to 3-KUDCA was carried out according to the following assay: individual colonies of recombinant P. pastoris strains were picked into 500 mL baffled conical flasks containing BMGY media (100 mL working volume). The flasks were incubated at 30oC for 72 h with shaking at 250 rpm at 85 % humidity and were supplemented with glycerol (2 % final concentration) every 24 hours. After this time, the biomass was harvested via centrifugation and the supernatant was discarded. The biomass was then resuspended in BMMY and incubated at 30oC with shaking at 250 rpm at 85 % humidity for 6 hours to induce protein production. After this time, the biomass was harvested via centrifugation and the supernatant removed. A portion of the resulting biomass (2 g) was resuspended in 10 mL of bioconversion buffer (295-10000 mg / L 3-KCA, 0.1 M phosphate buffer, 2 % MeOH, 2 mM 5-ALA, pH 9) and the flask was incubated at 30oC for 24 h with shaking at 250 rpm at 85 % humidity. The extraction, sample preparation and analytical methods are identical to those described in Example 1.

[0188] Results

[0189] Screening results for 3-KCA to 3-KUDCA bioconversion by the P. pastoris strain builds are summarized in Table 11 below. ‐ 84 ‐

[0190] TABLE 11 Strain 3-KCA Loading % Code P. pastoris Strain Description (mg / L) Conversionp g . p ost Cells by Site Saturation Mutagenesis (SSM)

[0191] This example illustrates the preparation of site saturation mutagenesis (SSM) libraries of engineered polypeptides derived from the parent CYP105 polypeptide of SEQ ID NO: 32 from Example 3 expressed in the SHS058 parent strain and screening these variant strains for improved activity in the bioconversion of 3-KCA to 3-KUDCA relative to the bioconversion of the parent strain SHS058.

[0192] Material and Methods

[0193] A. Site Saturation Mutagenesis library ( Round 3-SSM1-6 CYP105 backbone libraries) construction

[0194] The heterologous nucleic acid sequence from the single copy strain SHS058 build, which includes the codon-optimized polynucleotide sequence of SEQ ID NO:31 encoding the CYP polypeptide of SEQ ID NO: 32, expressed under the pGal1 promoter within the pGal1 / 10 promoter system (SEQ ID NO: 59) and PGK1t terminator (SEQ ID NO: 61), was used to design SSM oligonucleotide to generate SSM libraries at positions spanning the entire polypeptide sequence. To generate an appropriate screening strain to evaluate these libraries, the URA3 marker was integrated into the X-4 site (Easy-Clone 2.0) of a yeast strain under the pGal1 ‐ 85 ‐   promoter within the pGal1 / 10 promoter system (SEQ ID NO: 59) and PGKt1 terminator (SEQ ID NO: 61), along with the codon-optimized polynucleotide sequence of SEQ ID NO: 3, which encodes the CPR polypeptide of SEQ ID NO: 4, under the pGal10 promoter within the pGal1 / 10 promoter system (SEQ ID NO: 59) and ADH1t terminator (SEQ ID NO: 60). The resulting strain SHEVP003 was used as the negative control and screening host for CYP SSM library integration. Libraries were plated on selective media containing 5’-fluoroorotic acid, which selects for URA3 negative cells, indicating successful integration of DONOR DNA into the SHEVP003 screening strain. SHS058 strain was used as a control strain during screening of the SSM library strains for fold-improvement calculations with respect to conversion of 3-KCA to 3-KUDCA as described below.

[0195] Genomic DNA from the SHS058 strain was used as the template to generate two PCR products: (1) a first PCR product (Fragment A), which does not harbor any degenerate codons, and (2) a second PCR product (Fragment B), which has sequence overlap with the Fragment A, and is amplified harboring one NNK degenerate codon only. Primers spanning the full 513 codons of the CYP polypeptide were designed according to standard site-saturation mutagenesis protocols and used for amplification of Fragments A and B and overlap extension.

[0196] Fragment A was amplified using a single forward primer P34 (SEQ ID NO: 95) and a series of 513 reverse primers designed according to the location of the desired mutagenesis site in CYP. The 513 reverse primers for Fragment A listed in Table 6A (above) and are provided in the accompanying Sequence Listing as SEQ ID NOs: 102-614. Additional reverse primers were designed as to not perturb the codon mutations in the backbone of CYP polypeptide SEQ ID NO: 32. The reverse primers for Fragment A are provided in Table 12A below and are provided in the accompanying Sequence Listing as SEQ ID NOs: 1067-1114.

[0197] TABLE 12A Additional reverse primers for SSM library Fragment A amplification SEQ ID Primer Name Primer Sequence  NO: ‐ 86 ‐   Codon39_A2CTTGCCCCCGTTTCCGAGAAGAACATACTT1085Codon55_A6TTTATTCGACAGCTCAAAAGGTTTCTTAGG1086Codon57 A7AACTCGTTTATTCGACAGCTCAAAAGGTTT1087g p g p series of 513 forward primers that included a single NNK degenerate codon spanning across successive positions of the CYP encoding gene of SEQ ID NO: 31. The 513 forward primers for Fragment B are listed in Table 6B and are provided in the accompanying Sequence Listing as SEQ ID NOs: 615-984.. Additional forward primers were designed as to not perturb the codon mutations in the backbone of CYP polypeptide SEQ ID NO: 32. The forward primers for Fragment B are provided in Table 12B below and are provided in the accompanying Sequence Listing as SEQ ID NOs: 1115-1184.

[0199] TABLE 12B: Additional forward primers for SSM library Fragment B amplification SEQ ID Primer Name Primer Se uence NO:‐ 87 ‐ Codon310_E8CTGGTGGCGATTCACACAACAGCCGATTTANNKTCTGAAACAATG1123Codon314_E9CACACAACAGCCGATTTACTGTCTGAAACANNKTTTAATATAGCT1124Codon323 E10ACAATGTTTAATATAGCTTTGCAACCAGAANNKCTTGAGCCACTA1125‐   ‐   Codon63_A11AATAAACGAGTTGTCCAGGATTTTATCGAGNNKGCACGAGACATT1178Codon144_B11CCGCTCGTTAACAAGTATCTTACAAGGGGANNKGCAAAACTAACA1179Codon145 B12CTCGTTAACAAGTATCTTACAAGGGGATTGNNKAAACTAACAAAA1180primer P35 (SEQ ID NO: 96) and reverse primer of P36 (SEQ ID NO: 97). The assembled OE- PCR products were then pooled together, and gel purified to provide a saturation mutagenesis library of linear donor DNA. The pooled saturation mutagenesis library linear donor DNA was integrated as a knock-in using CRISPR-Cas9 into the X-4 locus where URA3 was integrated in SHEVP003 which already had the CPR gene integrated.

[0201] B. Screening of SSM library (R3)

[0202] Individual colonies of recombinant S. cerevisiae strains were picked into 96-well plates containing 2 x YPD media (300 ^L per well) using a QPixTM420 colony picking system. The plates were incubated at 30oC for 24 h with shaking at 250 rpm at 85 % humidity. After this time, the strains were sub-cultured (40 ^L inoculation volume per strain) into a second 96-well plate containing 1 x YPD media (300 ^L per well) using an Agilent Bravo automated liquid handling platform. The plates were incubated at 30oC for 48 h with shaking at 250 rpm at 85 % humidity. After this time, a solution of galactose (40 % stock solution) was added using the Bravo (1 % final concentration) and the plates were further incubated for 4 hours to induce protein production. After this time, the biomass was harvested via centrifugation and the supernatant removed. The resulting biomass was resuspended in 300 ^L of bioconversion buffer (590 mg / L 3-KCA, 0.1 M phosphate buffer, pH 9) using the Agilent Bravo automated liquid handling platform and the plates were incubated at 30oC for 24 h with shaking at 250 rpm at 85 % humidity. After this time, MeOH (300 ^L) was added using the Bravo and the plates were incubated at 30oC with shaking at 250 rpm at 85 % humidity for a further 30 minutes. The biomass was then separated by centrifugation and the resulting supernatant diluted with MeOH (1000 x total dilution) using the Agilent Bravo automated liquid handling platform before analysis using an Agilent 6470 Triple Quadrupole LC / MS.

[0203] LC / MS sample preparation: The bioconversion reaction was extracted and diluted with MeOH for sample preparation as described above. The prepared samples were loaded onto an Agilent 6470 Triple Quadrupole LC / MS and the compounds of interest were detected using MS / MS in SIM mode. The compounds were quantified relative to calibration curves which were prepared using serial dilutions of stock solutions of 3-KCA and 3-KUDCA.

[0204] LC / MS instrumentation and parameters: LC / MS system: Agilent 6470 Triple Quadrupole LC / MS; Column: Agilent EclipsePlusC18 column 1.8 um, 3.0 x 50 mm; Mobile phase: Solvent A: H2O with 0.1% formic acid; Solvent B: Acetonitrile with 0.1% formic acid; ‐ 89 ‐   Gradient: 0 - 0.2 min isocratic 40% B, 0.2 - 2.2 min gradient to 85% B, 2.2 - 2.8 min isocratic 85% B; Mode: MS / MS in SIM mode (monitoring at 389.3 for 3-KUDCA and 373.2 for 3-KCA); Fragmentor: 90 volts.

[0205] Results

[0206] Data from screening the SSM libraries in terms of fold-improvement in production of 3- KUDCA from 3-KCA relative to the control strain, SHS058 expressing CYP105 are summarized in Table 13 (below).

[0207] TABLE 13 AA differences relative 3-KCA to 3- FIOC 3-KCA to NT SEQ AA SEQ to CYP105 KUDCA % 3-KUDCA % N ID N ID N E ID N 2 i1i nExample 6: Further Optimization of Mutant CYP Genes in Recombinant P. pastoris Host Cells by Combinatorial Mutagenesis

[0208] This example illustrates the preparation of further combinatorial libraries of CYP genes based on the parent strain SHS088 which carries a gene of SEQ ID NO: 1187 encoding the CYP207 (SEQ ID NO: 1188) which has the 6 amino acid differences S31V, Q54K, A143G, T307A, G325E, V364L relative to the wild-type CYP of SEQ ID NO: 2, identified through SSM optimization of CYP105 for improved bioconversion of 3-KCA to 3-KUDCA described in Example 5.

[0209] Material and Methods

[0210] A. Combinatorial library construction (Round 4) ‐ 90 ‐

[0211] The polynucleotide sequence of SEQ ID NO: 1187, encoding polypeptide SEQ ID NO: 1188 (also referred to herein as “CYP207”) was used to design oligos to generate combinatorial libraries to randomly incorporate additional identified beneficial amino acid mutations from Example 5 into the CYP207 sequence. The same screening strain from Example 2, SHEVP003) was used as a host for integration of the resulting libraries. SHS088, which expresses the CYP207 polypeptide of SEQ ID NO: 1188 and the CPR001 polypeptide of SEQ ID NO: 4, was used as the control strain.

[0212] A semi-synthetic approach was used to construct the first set of combinatorial libraries. Genomic DNA from the SHS088 strain was used as the template to generate a full-length PCR product using primer pair of P34 (SEQ ID NO: 95) and P37 (SEQ ID NO: 98), while incorporating uracil using a dNTP mix comprising of the following deoxyribonucleotides: dATP, dGTP, dCTP, dTTP, dUTP. The resulting PCR product was gel purified and digested with Uracil-DNA Glycosylase and Endonuclease IV at 37 C for 2 hours, followed by enzyme denaturation at 94 C for two minutes, to generate a pool of fragments in the range of 50-100 bases. These fragments were further combined with differing ratios of pools of the synthesized oligonucleotide primers (each oligo up to 55 bases in length and encoding one or more amino acid change) in several individual assembly PCR reactions using forward primer P5 (SEQ ID NO: 66) and reverse primer P6 (SEQ ID NO: 67) to reassemble the full-length PCR product as Fragment B and incorporate mutagenic amino acid changes within each pool randomly. The synthesized oligonucleotide primers SEQ ID NOs: 1221-1242 used in the pools and their encoded mutations are listed in Table 14 below and the accompanying Sequence Listing.

[0213] TABLE 14 SEQ ID Primer Name Primer Sequence NO:‐ 91 ‐   CACTACGTGAAGAGATAGTTGTCGTTTTATCCACGGAAGGTCTAAAA 1234 ELITE2_T333V AAGACGTCGTT CACTACGTGAAGAGATAGTTACGGTTTTATCCACGCAAGGTCTAAAA 1235h homologous 5’ and 3’ regions to the CYP were generated (Fragment A and Fragment C respectively). Fragment A was amplified using the forward primer P37 (SEQ ID NO: 98) and reverse primer P4 (SEQ ID NO: 65) and Fragment C was amplified using the forward primer P7 (SEQ ID NO: 68) and the reverse primer P38 (SEQ ID NO: 99). Fragments A, B, and C were assembled by NEBuilder® HiFi Assembly and amplified as a single linear DONOR using the following forward rescue primer P35 (SEQ ID NO: 96) and reverse primer P36 (SEQ ID NO: 97). The assembled PCR products were then pooled together, and gel purified to provide a combinatorial library of linear donor DNA.

[0215] The pooled combinatorial libraries of linear donor DNA were integrated as a knock-in using CRISPR-Cas9 into the X-4 locus where URA3 was integrated in SHEVP003 which already had the CPR gene integrated as in Examples 1 and 2.

[0216] B. Screening of Combi library

[0217] Screening of the recombinant host cells for bioconversion of 3-KCA to 3-KUDCA was carried out using the same protocol described for screening the SSM library except 0.5 x YPD was used for growth and 3-KCA loading was increased from 590 mg / L to 800 mg / L.

[0218] Results

[0219] Screening of the combinatorial libraries for fold-improvement in production of 3-KUDCA from 3-KCA relative to the control strain, SHS088, are summarized in Table 15 (below).

[0220] TABLE 15 3-KCA to 3- FIOC 3-KCA NT SEQ AA SEQ AA differences relative to KUDCA % to 3-KUDCA n       1257 1258 K70L, E338Q, M485T 19.5 0.9 1259 1260 N316S 19.5 0.9pastoris Host Cells

[0221] This example illustrates the preparation of recombinant yeast host cell strains (Pichia pastoris) with genomically integrated copies of a pair of heterologous genes encoding a cytochrome P450 (CYP) and cytochrome reductase (CPR) from Gibberella zeae and demonstration of the ability of these strains to carry out the bioconversion of Lithocholic acid (LCA) to Ursodeoxycholic acid (UDCA) and / or 3-Keto-lithocholic acid (3-KCA) to 3-Keto- ursodeoxycholic acid (3-KUDCA).

[0222] Material and Methods

[0223] A. P. pastoris strain building

[0224] A recombinant Pichia host cell strain (SHP070) with one copy of the gene encoding CYP110 (SEQ ID NO: 1185) was also constructed as follows. A single plasmid, wbplasmid226 (SEQ ID NO: 1273) containing a geneticin resistance marker, an ampicillin resistance marker, a pUC origin of replication, and a cassette comprising of pAOX1:CYP110:tDAS2 was assembled as described above. The wbplasmid226 shares the same sequence as wbplasmid202 (SEQ ID NO: 1066) except for the CYP105 coding sequence (SEQ ID NO: 31) was replaced with the CYP110 coding sequence of SEQ ID NO: 1185. Inverse PCR was used to amplify the vector minus the CYP100 coding sequence using primers wboligos5803 and wboligos5921. The gene encoding CYP110 (SEQ ID NO: 1185) was amplified from SHS062 gDNA using primers wboligos5922 and wboligos5802. CYP110 in SHS062 was expressed under the pGal1 promoter within the pGal1 / 10 promoter system (SEQ ID NO: 59) and PGK1t terminator (SEQ ID NO: 61). Following plasmid recovery and sequence confirmation as described above, a single plasmid was digested with SacI and further purified using Zymo Research’s DNA Clean and Concentrator Kit (catalog no. D4014). The restriction enzyme cut site, found in pAOX1, linearized the plasmid, allowing for homologous integration at AOX1 locus. Five microliters of linearized plasmid were used to transform 50 μL of BG-11 competent cells and plated on YPD media plates containing geneticin. After 3 days of growth, individual colonies were confirmed for integration of the linearized plasmid using PCR and genomic DNA from each individual colony.

[0225] A recombinant Pichia host cell strain (SHP079) with one copy of the gene encoding CYP207 (SEQ ID NO: 1187) was also constructed as follows. A single plasmid, wbplasmid231 (SEQ ID NO: 1274), containing a geneticin resistance marker, an ampicillin resistance marker, ‐ 93 ‐   a pUC origin of replication, and a cassette comprising of pAOX1:CYP207:tDAS2 was assembled as described above. The wbplasmid231 shares the same sequence as wbplasmid202 (SEQ ID NO: 1066) except for the CYP105 coding sequence (SEQ ID NO: 31) was replaced with the CYP207 coding sequence of SEQ ID NO: 1187. Inverse PCR was used to amplify the vector minus the CYP100 coding sequence using primers wboligos5803 and wboligos5921. The gene encoding CYP207 (SEQ ID NO: 1187) was amplified from SHS088 gDNA using primers wboligos5922 and wboligos5802. Following plasmid recovery and sequence confirmation as described above, a single plasmid was digested with SacI and further purified using Zymo Research’s DNA Clean and Concentrator Kit (catalog no. D4014). The restriction enzyme cut site, found in pAOX1, linearized the plasmid, allowing for homologous integration at AOX1 locus. Five microliters of linearized plasmid were used to transform 50 μL of BG-11 competent cells and plated on YPD media plates containing geneticin. After 3 days of growth, individual colonies were confirmed for integration of the linearized plasmid using PCR and genomic DNA from each individual colony.

[0226] A recombinant Pichia host cell strain (SHP083) with one copy of the gene encoding CYP110 (SEQ ID NO: 1185) was also constructed as follows. A single plasmid, wbplasmid226, was digested with SacI and further purified using Zymo Research’s DNA Clean and Concentrator Kit (catalog no. D4014). The restriction enzyme cut site, found in pAOX1, linearized the plasmid, allowing for homologous integration at AOX1 locus. Five microliters of linearized plasmid were used to transform 50 μL of BG-45 competent cells (a double knockout host strain; aox1-KO, yps1-KO) and plated on YPD media plates containing geneticin. After 3 days of growth, individual colonies were confirmed for integration of the linearized plasmid using PCR and genomic DNA from each individual colony. The strain build lineage for the above-described SHP083 P. pastoris strain is summarized in the chart of FIG.4.

[0227] B. Screening of P. pastoris strains for 3-KCA to 3-KUDCA conversion

[0228] Screening of the recombinant host cells for bioconversion of 3-KCA to 3-KUDCA (as shown in Scheme 1) was carried out according to the following assay: individual colonies of recombinant P. pastoris strains were picked into 500 mL baffled conical flasks containing BMGY media (100 mL working volume). The flasks were incubated at 30oC for 48 h with shaking at 250 rpm at 85 % humidity and were supplemented with glycerol (1 % final concentration) every 24 hours. After this time, the biomass was harvested via centrifugation and the supernatant was discarded. A portion of the resulting biomass (0.5 g) was resuspended in 2.5 mL of bioconversion buffer (10-20 g / L 3-KCA, 0.1 M phosphate buffer, 2 % MeOH pH 9) and the vial was incubated at 30oC for 72 h with shaking at 250 rpm at 85 % humidity. One volume (2.5 mL) of methanol was then added to the vial for extraction with shaking at 30oC for 30 mins. The supernatant was then collected via centrifugation and diluted / analyzed as described in previous examples.

[0229] Screening of P. pastoris strains for DCA to UCA and CA conversion  ‐ 94 ‐

[0230] Screening of the recombinant host cells for bioconversion of DCA to UCA / CA (as shown in Scheme 2) was carried out according to the following assay: individual colonies of recombinant SHP070 were picked into 500 mL baffled conical flasks containing BMGY media (100 mL working volume). The flasks were incubated at 30oC for 48 h with shaking at 250 rpm at 85 % humidity and were supplemented with glycerol (1 % final concentration) every 24 hours. After this time, the biomass was harvested via centrifugation and the supernatant was discarded. A portion of the resulting biomass (0.5 g) was resuspended in 2.5 mL of bioconversion buffer (2 g / L DCA, 0.1 M phosphate buffer, 2 % MeOH pH 9) and the vial was incubated at 30oC for 72 h with shaking at 250 rpm at 85 % humidity. One volume (2.5 mL) of methanol was then added to the vial for extraction with shaking at 30oC for 30 mins. The supernatant was then collected via centrifugation and diluted as described in previous examples. The below LC / MS method was used to confirm the production of UCA (23 % conversion) and CA (0.1 % conversion).

[0231] LC / MS instrumentation and parameters: LC / MS system: Agilent 6470 Triple Quadrupole LC / MS; Column: Agilent EclipsePlusC18 column 1.8 um, 3.0 x 50 mm; Mobile phase: Solvent A: H2O with 0.1% formic acid; Solvent B: Acetonitrile with 0.1% formic acid; Gradient: 0 - 0.2 min isocratic 40% B, 0.2 - 2.2 min gradient to 85% B, 2.2 - 2.8 min isocratic 85% B; Mode: MS / MS in SIM mode (monitoring at 391.5 for DCA and 407.5 for UCA and CA); Fragmentor: 90 volts.

[0232] Results

[0233] Screening results for 3-KCA to 3-KUDCA bioconversion by the P. pastoris strain builds are summarized in Table 16 below.

[0234] TABLE 16 Strain 3-KCA Loading % Code P pastoris Strain Description (mg / L) Conversion

[0235] Screening results for DCA to UCA / CA bioconversion by the P. pastoris strain builds are summarized in Table 17 below.

[0236] TABLE 17 DCA n‐   ‐   Example 8: Design and Expression of a Heterologous CYP-CPR Fusion Polypeptide in Pichia pastoris Host Cells

[0237] This example illustrates the design and expression of a heterologous gene encoding a fusion of a cytochrome P450 (CYP) from Gibberella zeae and an endogenous cytochrome reductase (CPR) from Pichia. The resulting cytosolic fused enzyme with CYP and CPR activities demonstrated an ability to carry out the bioconversion of Lithocholic acid (LCA) to Ursodeoxycholic acid (UDCA) and / or 3-Keto-lithocholic acid (3-KCA) to 3-Keto-ursodeoxycholic acid (3-KUDCA).

[0238] Materials and Methods

[0239] A. Design CYP207-CPR fusion

[0240] A polynucleotide of SEQ ID NO: 1275 was synthesized that encodes an N-terminal 31 amino acid truncation of CYP207 named trCYP207 (SEQ ID NO:1276) fused via short polypeptide linker (aa sequence: ARA) to the N-terminus of a truncated version of the Pichia pastoris endogenous reductase, trNCP1 (SEQ ID NO: 1278). The resulting cytosolic fused enzyme encoded by the fusion gene of SEQ ID NO: 1279 has the amino acid sequence of SEQ ID NO: 1280. The fusion and its various constituent polynucleotide and polypeptide sequences are provided in Table 18 below and the accompanying Sequence Listing.

[0241] TABLE 18 SEQ ID Description NO: Sequence A C C A C T A A G G G A G T A C T G A G T C G T C C T‐   ‐ ATGCTATTGAAATATGATTGGAAATTACCAGAAGGTGTTGTACCTAAG TCTAAGGCTTTAGGAATGTCTTTACTTGGTGACCGGGAAGCCAAACTG ATGGTGAAAAGGAGAGCAGCCGAAATCGATATAGACACTATTGGTAGT P G G P P L G F H S G C G A G A C T G C T A A T T A C T C A C T G C T C C G C A T T G T A C A C T A T‐ 97 ‐ trNCP11278GKEDDNSVHGVAGGFQTRDLVEILNSTNKKALVLYGSQTGTSEDYAHKpolypeptide YARELQSKFSIPTLCGDLSEFDFDNLNDIPEQVEGFTFITFFMATYGE GEPTDNAVEFIEFLKNDAEDLSNLKYTVFGLGNSTYEFYNQMGKTTNK E T N H N I R Q F I A C C A C T A A G G G A G T A C T G A G T C G T C C T G G T A C G T C G C T T A     ACCTTTGGCGAGGGCGATGATGGTCAGGCAACTATGGACGAGGACTTC CTAGCATGGAAAGATTCCCTGTTTGATACAATCAAGAAGGATTTGCAT TTGGAGGAACATGAAGTTGTCTACCAGCCAGGTCTAAAAGTAAAGGAG T T A T C G G A A G A A T G A T T T G C A C G G A T A A P G G P P L G F H S E M G H G A A T K D I K D‐ 99 ‐

[0242] The trCYP207-trNCP1 fusion gene of SEQ ID NO: 1280 was constructed for transformation in Pichia under the pAOX1 promoter and DAS2 terminator sequences expressed at the pAOX1 region of Pichia pastoris strain BG-45 ( ^yps, ^aox1). Transformation of Pichia was carried library style and named libSHP153.

[0243] Screening of the libSHP153 library for recombinant host cells capable of the bioconversion of 3-KCA to 3-KUDCA was carried out as described in Example 7. SHP089 (CYP207 @AOX1) was used as control strain and showed 53.6 % (+ / -4.5) bioconversion of 3- KCA to 3-KUDCA.

[0244] Results

[0245] As shown by the plot of screening results depicted in FIG.5, libSHP153 clone_E3 showed the highest conversion, 76.9 % or 1.4 FIOPC (SHP089) and promoted to strain SHP132. A genotype summary of strain SHP132 is depicted in FIG.4. Example 9: Design of Recombinant Pichia pastoris Host Cells that Overexpress Endogenous NCP1 Gene with CPR Activity

[0246] This example illustrates the preparation of a recombinant Pichia host cell that heterologously expresses the engineered CYP110 polypeptide (SEQ ID NO: 1186) with CYP activity and overexpresses the endogenous cytochrome reductase NCP1 gene with CPR activity from Pichia. The resulting recombinant host cells overexpresses the endogenous CPR activity and demonstrated an ability to carry out the bioconversion of Lithocholic acid (LCA) to Ursodeoxycholic acid (UDCA) and / or 3-Keto-lithocholic acid (3-KCA) to 3-Keto-ursodeoxycholic acid (3-KUDCA).

[0247] Materials and Methods

[0248] A. Design of engineered Pichia strain

[0249] The SHP083 strain of Example 7 was used as the parent strain to integrate an extra copy of the PpNCP1 gene with CPR activity into the His4 locus under the control of pAOX1 and tDAS1 terminator. The transformation was carried out library style and named libSHP155. See Figure 2 for a summary of the results.

[0250] Screening of the libSHP155 library for recombinant host cells capable of the bioconversion of 3-KCA to 3-KUDCA was carried out as described in Example 7. SHP083 was used as control strain and showed around 55 % bioconversion of 3-KCA to 3-KUDCA.

[0251] Results

[0252] As shown by the plot of screening results depicted in FIG.6, the libSHP155_C3 clone showed the highest conversion, 100% (or 1.5 FIOPC). The top four clones were promoted to strains SHP143, SHP144, SHP145 and SHP146. A genotype summary of these four strains is shown in FIG.4. ‐ 100 ‐

[0253] While the foregoing disclosure of the present invention has been described in some detail by way of example and illustration for purposes of clarity and understanding, this disclosure including the examples, descriptions, and embodiments described herein are for illustrative purposes, are intended to be exemplary, and should not be construed as limiting the present disclosure. It will be clear to one skilled in the art that various modifications or changes to the examples, descriptions, and embodiments described herein can be made and are to be included within the spirit and purview of this disclosure and the appended claims. Further, one of skill in the art will recognize a number of equivalent methods and procedure to those described herein. All such equivalents are to be understood to be within the scope of the present disclosure and are covered by the appended claims.

[0254] Additional embodiments of the invention are set forth in the following claims.

[0255] The disclosures of all publications, patent applications, patents, or other documents mentioned herein are expressly incorporated by reference in their entirety for all purposes to the same extent as if each such individual publication, patent, patent application or other document were individually specifically indicated to be incorporated by reference herein in its entirety for all purposes and were set forth in its entirety herein. In case of conflict, the present specification, including specified terms, will control.      ‐ 101 ‐

Claims

CLAIMS 1. A recombinant host cell comprising a heterologous nucleic acid encoding a polypeptide with CYP activity capable of converting LCA to UDCA and / or converting 3-KCA to 3-KUDCA, wherein the polypeptide with CYP activity comprises an amino acid sequence at least 80% sequence identity to SEQ ID NO: 2 and an amino acid difference relative to SEQ ID NO: 2 at one or more positions selected from L7, S31, Q54, V56, K70, K76, I120, A143, L147, A153, N160, H170, G175, E176, V210, V261, T307, N316, Q320, G325, T333, E338, V364, L365, P401, E402, S408, Q424, L444, F452, K479, and M485.

2. The cell of claim 1, wherein the amino acid differences are selected from L7R, S31V, Q54K, V56G, K70L, K70N, K76H, I120A, A143G, A143R, L147R, L147W, A153Q, A153R, N160H, H170V, G175E, E176D, V210R, V261E, T307A, N316S, Q320M, G325E, T333V, E338Q, V364L, L365A, P401D, E402V, S408R, Q424V, L444F, F452D, K479R, and M485T.

3. The cell of any one of claims 1-2, wherein the polypeptide amino acid sequence comprises a combination of amino acid differences selected from: S31V, Q54K, A143G, T307A, G325E S31V, Q54K, A143G, T307A, G325E, L365A‐ 102 ‐   L7R, S31V, Q54K, A143G, V210R, T307A, G325E, V364L, K479R L7R, S31V, Q54K, K70L, A143G, N160H, H170V, E176D, T307A, G325E, V364L 4. Thece o a y o e o ca s - , e e e eeoogous ucec ac e co g e polypeptide further comprises a silent mutation selected from R328 (CGT > CGC), A368 (GCT > GCC), and V476 (GTT > GTC).

5. The cell of any one of claims 1-4, wherein the heterologous nucleic acid comprises: (a) a sequence of at least 80% identity to a sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 1185, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267, 1269, 1271, and 1275; or (b) a codon degenerate sequence of a sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 1185, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267, 1269, 1271, and 1275.

6. The cell of any one of claims 1-5, wherein the polypeptide with CYP activity comprises an amino acid sequence having at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO:

2.

7. The cell of any one of claims 1-6, wherein the polypeptide with CYP activity comprises an amino acid sequence selected from SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1244, ‐ 103 ‐   1246, 1248, 1250, 1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, and 1272; optionally, wherein the polypeptide is truncated by 1-31 amino acids at its N-terminus.

8. The cell of any one of claims 1-7, wherein the heterologous nucleic acid encodes a polypeptide with CPR activity; optionally, wherein the polypeptide with CPR activity comprises an amino acid sequence of at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 4 and 1278.

9. The cell of any one of claims 1-8, wherein the heterologous nucleic acid encodes a polypeptide with CYP activity fused via a linker to a second polypeptide with CPR activity; optionally, wherein the heterologous nucleic acid encodes a polypeptide comprising an amino acid sequence of at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 1280.

10. The cell of any one of claims 1-9, wherein the heterologous nucleic acid is integrated into a site in the host cell genome; optionally integrated at two or more sites in the genome.

11. The cell of any one of claims 1-10, wherein the heterologous nucleic acid is under the control of a promoter system selected from pGal1 / 10, and pCAT1:pFDH1.

12. The cell of any one of claims 1-11, wherein the source organism of the recombinant host cell is selected from Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, and Escherichia coli.

13. The cell of any one of claims 1-12, wherein the recombinant host cell is Saccharomyces cerevisiae and the heterologous nucleic acid is integrated at one or more sites in the genome selected from X-4, XI-2, XII-4, and X-2.

14. The cell of claim 13, wherein the heterologous nucleic acid is integrated at three or more sites; optionally, integrated at four or more sites.

15. The cell of any one of claims 1-12, wherein the recombinant host cell is Pichia pastoris and the heterologous nucleic acid is integrated at one or more sites in the genome selected from AOX1, Int6, Int15, and HIS4. ‐ 104 ‐   16. The cell of claim 15, wherein the heterologous nucleic acid is integrated at three or more sites; optionally, integrated at four or more sites.

17. A method for producing UDCA or 3-KUDCA comprising: (a) culturing a recombinant host cell of any one of claims 1-16 in a suitable medium comprising LCA and / or 3-KCA; and (b) recovering the produced UDCA and / or 3-KUDCA.

18. A recombinant polypeptide with CYP activity capable of converting LCA to UDCA and / or converting 3-KCA to 3-KUDCA, wherein the polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2 and an amino acid difference relative to SEQ ID NO: 2 at one or more positions selected from L7, S31, Q54, V56, K70, K76, I120, A143, L147, A153, N160, H170, G175, E176, V210, V261, T307, N316, Q320, G325, T333, E338, V364, L365, P401, E402, S408, Q424, L444, F452, K479, and M485.

19. The polypeptide of claim 18, wherein the amino acid differences are selected from L7R, S31V, Q54K, V56G, K70L, K70N, K76H, I120A, A143G, A143R, L147R, L147W, A153Q, A153R, N160H, H170V, G175E, E176D, V210R, V261E, T307A, N316S, Q320M, G325E, T333V, E338Q, V364L, L365A, P401D, E402V, S408R, Q424V, L444F, F452D, K479R, and M485T.

20. The polypeptide of any one of claims 18-19, wherein the polypeptide amino acid sequence comprises a combination of amino acid differences selected from: S31V, Q54K, A143G, T307A, G325E S31V, Q54K, A143G, T307A, G325E, L365A‐   ‐   S31V, Q54K, A143G, T307A, G325E, E338Q S31V, Q54K, K70N, A143G, T307A, G325E, S31V Q54K A143G V210R T307A G325E L 21. Thepolypeptide of any one of claims 18-20, wherein the polypeptide comprises an amino acid sequence having at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO:

2.

22. The polypeptide of any one of claims 18-21, wherein the polypeptide comprises an amino acid sequence selected from SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1244, 1246, 1248, 1250, 1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, 1272, and 1276.

23. The polypeptide of any one of claims 18-22, wherein the polypeptide comprises an N- terminal truncation of from 1 to 31 amino acids as compared to SEQ ID NO: 2; optionally, wherein the polypeptide comprises an amino acid of SEQ ID NO: 1276.

24. The polypeptide of any one of claims 18-23, wherein the polypeptide is fused via a linker to a second polypeptide; optionally, wherein the second polypeptide has CPR activity.

25. The polypeptide of claim 24, wherein the second polypeptide with CPR activity comprises an amino acid sequence having at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least ‐ 106 ‐   91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 4 and 1278; optionally, wherein the polypeptide comprises an amino acid sequence of SEQ ID NO: 1280.

26. A polynucleotide encoding the polypeptide of any one of claims 18-22.

27. The polynucleotide of claim 26 in which the polynucleotide sequence comprises: (a) a sequence of at least 80% identity to a sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 1185, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267, 1269, 1271, and 1275; or (b) a codon degenerate sequence of a sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 1185, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267, 1269, 1271, and 1275.

28. An expression vector comprising the polynucleotide of any one of claims 26-27.

29. The expression vector of claim 28 comprising a control sequence.

30. A host cell comprising the polynucleotide of any one of claims 26-27 or the expression vector of any one of claims 28-29.

31. A method for preparing a compound of structural formula (I) wherein,R1is hydrogen, a carboxylate salt counterion, an optionally substituted C1-C20 alkyl, or an optionally substituted aryl; R2is hydrogen, or a hydroxyl; R3is a hydroxyl, or an oxo group; ‐ 107 ‐   the method comprising contacting under suitable reactions conditions a compound of structural formula (II) wherein, R1issubstituted C1-C20 alkyl, or an optionally substituted aryl; R2is hydrogen, or a hydroxyl; R3is a hydroxyl, or an oxo group; and a recombinant polypeptide of any one of claims 18-22.

32. The method of claim 31, wherein R2is hydrogen.

33. The method of claim 32, wherein: (a) the compound of structure formula (I) is UDCA and the compound of structural formula (II) is LCA; or (b) the compound of structure formula (I) is 3-KUDCA and the compound of structural formula (II) is 3-KCA.

34. The method of claim 31, wherein R2is a hydroxyl.

35. The method of claim 34, wherein the compound of structure formula (I) is UCA and the compound of structural formula (II) is DCA.     ‐ 108 ‐

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