Modified microorganisms for fermentative production of progesterone and ursodeoxycholic acid

Abstract

The present disclosure relates to genetically engineered microorganisms for producing progesterone and / or ursodeoxycholic acid by fermentation.

Description

[0001] Priority claim

[0002] This application claims the benefit of International Application No. PCT / CN2022 / 141263, filed on December 23, 2022, the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically as an XML file named "50548-0005WO2_SL_ST26".XML. This XML file was created on December 13, 2023, and is 190,216 bytes in size. The material in the XML file is incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure relates to genetically engineered microorganisms for producing progesterone and / or ursodeoxycholic acid by fermentation. The present disclosure also relates to genetically engineered microorganisms for producing 4-cholic acid-3-one, 3-keto-lithocholic acid, and lithocholic acid. Background Art

[0006] Progesterone is a naturally occurring steroid produced in the human body and an active pharmaceutical ingredient (API) commonly used in female hormone replacement therapy. Ursodeoxycholic acid is a drug used to manage and treat cholestatic liver disease. Most current production methods utilize chemical steps that require harsh chemicals and solvents. Therefore, biosynthesis and / or biotransformation are considered alternatives with high yields and less chemical waste. Summary of the Invention

[0007] The present disclosure relates to modified microbial cells, cultures of microbial cells, and methods for producing progesterone and / or ursodeoxycholic acid (UDCA). The present disclosure also relates to genetically modified microorganisms for producing 4-cholic acid-3-one, 3-keto-lithocholic acid, and lithocholic acid.

[0008] In one aspect, the present disclosure relates to a modified microbial cell that produces progesterone, wherein in certain embodiments, the modified microbial cell expresses: (a) a non-native side chain cleavage P450 enzyme; and (b) one or more non-native redox chaperone proteins. In certain embodiments, the one or more non-native redox chaperone proteins comprise a non-native ferredoxin and / or a non-native ferredoxin reductase. In certain embodiments, the non-native side chain cleavage P450 enzyme has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: 1-4 and 7-24. In certain embodiments, the non-native ferredoxin protein has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to any one of SEQ ID NOs: 5, 25-37, and 137. In certain embodiments, the non-native ferredoxin reductase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to any one of SEQ ID NOs: 6, 38-46, and 136. In certain embodiments, the non-native side-chain cleavage P450 enzyme has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a Novosphingobium sp. PP1Y side-chain cleavage P450 enzyme (e.g., SEQ ID NO: 18) or a Novosphingobium subterraneum side-chain cleavage P450 enzyme (e.g., SEQ ID NO: 3).

[0009] In certain embodiments, (a) the non-native ferredoxin has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a Synechococcus elongatus ferredoxin (e.g., SEQ ID NO: 33); and the non-native ferredoxin reductase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a Synechococcus elongatus ferredoxin reductase (e.g., SEQ ID NO: 45); (b) the non-native ferredoxin has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a Chlamydomonas reinhardtii ferredoxin (e.g., SEQ ID NO: NO:29); and the non-native ferredoxin reductase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a Synechococcus elongatus ferredoxin reductase (e.g., SEQ ID NO:43); or (c) the non-native ferredoxin reductase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a Spinacia oleracea ferredoxin reductase (e.g., SEQ ID NO: NO:5) has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity; and the non-native state ferredoxin reductase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a Spinacia oleracea ferredoxin reductase (e.g., SEQ ID NO:6).

[0010] In certain embodiments, the non-native state side-chain cleavage P450 enzyme has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a Novosphingobium sp. PP1Y side-chain cleavage P450 enzyme (e.g., SEQ ID NO: 18); in certain embodiments, the non-native state ferredoxin has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a Spinacia oleracea ferredoxin (e.g., SEQ ID NO: 5); in certain embodiments, the non-native state ferredoxin reductase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a Spinacia oleracea ferredoxin reductase (e.g., SEQ ID NO: 6). NO:6) has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity.

[0011] In certain embodiments, the modified microbial cell comprises an expression vector comprising: a first gene sequence encoding the non-native side chain cleavage P450 enzyme, and a second gene sequence encoding the non-native ferredoxin or non-native ferredoxin reductase. In certain embodiments, the modified microbial cell comprises an expression vector comprising: a first gene sequence encoding the non-native side chain cleavage P450 enzyme, a second gene sequence encoding the non-native ferredoxin, and a third gene sequence encoding the non-native ferredoxin reductase. In certain embodiments, the expression vector further comprises a promoter (e.g., a constitutive promoter). In certain embodiments, the promoter is Ptac or Ppms. In certain embodiments, the expression vector further comprises a ribosome binding site upstream of the first gene sequence, the second gene sequence, and / or the third gene sequence.

[0012] In certain embodiments, two of the first gene sequence, the second gene sequence, and the third gene sequence are linked to express a fusion protein. In certain embodiments, the fusion protein comprises a connecting peptide sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 54 or 55. In certain embodiments, the fusion protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 47-53.

[0013] In certain embodiments, the expression vector comprises, from 5' to 3' end: (a) a second gene sequence encoding a non-native ferredoxin having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a Spinacia oleracea ferredoxin (e.g., SEQ ID NO: 5), (b) a first gene sequence encoding a non-native side-chain cleavage P450 enzyme having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a Novosphingobium sp. PP1Y side-chain cleavage P450 enzyme (e.g., SEQ ID NO: 6), NO:18) having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity, and (c) a third gene sequence encoding a non-native ferredoxin reductase having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a spinach (Spinacia oleracea) ferredoxin reductase (e.g., SEQ ID NO:6); in certain embodiments, the non-native ferredoxin and the non-native side-chain cleavage P450 enzyme are fused via a linker peptide having the sequence set forth in SEQ ID NO:54.

[0014] In certain embodiments, the expression vector comprises, from the 5' end to the 3' end: (a) a first gene sequence encoding a non-native side-chain cleavage P450 enzyme having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a Novosphingobium sp. PP1Y side-chain cleavage P450 enzyme (e.g., SEQ ID NO: 18), (b) a second gene sequence encoding a non-native ferredoxin protein having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a Novosphingobium sp. PP1Y side-chain cleavage P450 enzyme (e.g., SEQ ID NO: 18), and (b) a second gene sequence encoding a non-native ferredoxin protein having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a Spinacia oleracea ferredoxin protein (e.g., SEQ ID NO: NO:5) having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity, and (c) a third gene sequence encoding a non-native ferredoxin reductase having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a Spinacia oleracea ferredoxin reductase (e.g., SEQ ID NO:6); in certain embodiments, the non-native side-chain cleavage P450 enzyme and the non-native ferredoxin are fused via a linker peptide having the sequence set forth in SEQ ID NO:54.

[0015] In certain embodiments, the expression vector comprises, from the 5' end to the 3' end: (a) a first gene sequence encoding a non-native side-chain cleavage P450 enzyme having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a Novosphingobium sp. PP1Y side-chain cleavage P450 enzyme (e.g., SEQ ID NO: 18), (b) a second gene sequence encoding a non-native ferredoxin protein having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a Novosphingobium sp. PP1Y side-chain cleavage P450 enzyme (e.g., SEQ ID NO: 18), and (b) a second gene sequence encoding a non-native ferredoxin protein having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a Spinacia oleracea ferredoxin protein (e.g., SEQ ID NO: NO:5) having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity, and (c) a third gene sequence encoding a non-native ferredoxin reductase having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to a Spinacia oleracea ferredoxin reductase (e.g., SEQ ID NO:6); in certain embodiments, the non-native side-chain cleavage P450 enzyme and the non-native ferredoxin are fused via a linker peptide having the sequence set forth in SEQ ID NO:55.

[0016] In certain embodiments, the transformed microbial cells comprise one or more proteins involved in plant sterol degradation whose activity is reduced, and in certain embodiments, the activity of the reduction refers to a reduction relative to a control cell. In certain embodiments, the one or more proteins involved in plant sterol degradation are selected from steroid C27-monooxygenase, cytochrome P450, 3-ketosteroid-δ-1-dehydrogenase and / or 3-α, 7-α, 12-α-trihydroxy-5-β-cholesterol-24-enoyl-CoA hydratase. In certain embodiments, the activity of the reduction is achieved by one or more of the following means: gene deletion (e.g., gene knockout), gene disruption, altering the regulation of a gene, replacing a native promoter with a less active promoter, random mutagenesis, knockout based on the full genome scope of CRISPR, and expression of a protein or protein variant with reduced activity. In certain embodiments, the engineered microbial cell comprises one or more gene knockouts comprising: (a) smo3; (b) cyp142, smo3, and smo1; (c) cyp142, smo3, smo2, and kstD; (d) cyp142, smo3, smo2, chsH3, and kstD; (e) cyp142, smo3, smo2, and smo1; or (f) cyp142, smo3, smo2, smo1, and smo4.

[0017] In certain embodiments, the engineered microbial cell further expresses cholesterol oxidase, hydroxysteroid dehydrogenase, and / or ketosteroid isomerase.

[0018] In one aspect, the present disclosure relates to a method of culturing the engineered microbial cells described herein, comprising culturing the cells under conditions suitable for producing progesterone (e.g., in the presence of a phytosterol (e.g., β-sitosterol, β-sitostanol, campesterol, stigmasterol and / or brassicasterol)), optionally, in certain embodiments, further comprising recovering the progesterone from the culture.

[0019] On the one hand, the present disclosure relates to a modified microbial cell that produces 4-cholic acid-3-one (4CA3O), 3-keto-lithocholic acid (3-keto-LCA), lithocholic acid (LCA) and / or ursodeoxycholic acid (UDCA). In certain embodiments, the modified microbial cell comprises one or more proteins involved in plant sterol degradation with reduced activity, and in certain embodiments, the reduced activity refers to a reduction relative to a control cell. In certain embodiments, the one or more proteins involved in plant sterol degradation include acyl-CoA dehydrogenase, 3-α,7-α,12-α-trihydroxy-5-β-chole-24-enoyl-CoA hydratase, and / or 3-ketoacyl-ACP reductase. In certain embodiments, the reduced activity is achieved by one or more of the following means: gene deletion (e.g., gene knockout), gene disruption, altering the regulation of a gene, replacing a native promoter with a less active promoter, random mutagenesis, genome-wide knockout based on CRISPR, and expression of a protein or protein variant with reduced activity. In certain embodiments, the engineered microbial cell comprises one or more gene knockouts comprising: (a) casC3; (b) chsE3, chsE1, and chsE2; (c) chsE3, chsE1, chsE2, and casC; (d) chsE3, chsE1, chsE2, and chsH3; (e) chsE3, chsE1, chsE2, and hsd4A; (f) chsE3, chsE1, chsE2, and hsd4A; sE2 and casC3; (g) chsE3, chsE1, chsE2, casC3 and chsH3; (h) chsE3, chsE1, chsE2, casC3, chsH3 and chsE5; (i) chsE3, chsE1, chsE2, casC3, chsH3 and chsE4; or (j) chsE3, chsE1, chsE2, casC3, chsH3, chsE4 and chsE5.

[0020] In certain embodiments, the modified microbial cell further comprises one or more upstream enzymes that increase the activity of 4-cholic acid-3-one (4CA3O). In certain embodiments, the increased activity refers to an increase relative to a control cell. In certain embodiments, the one or more upstream enzymes include a thioesterase. In certain embodiments, the activity of one or more upstream pathway enzymes is increased by expressing an enzyme or enzyme variant that has an increased expression level relative to the native enzyme.

[0021] In certain embodiments, the modified microbial cell further comprises one or more proteins involved in 4CA3O degradation with reduced activity. In certain embodiments, the reduced activity refers to a reduction relative to a control cell. In certain embodiments, the one or more proteins involved in 4CA3O degradation are selected from acyl-CoA synthetase, acyl-CoA ligase, acyl-CoA reductase, and aldehyde reductase. In certain embodiments, the gene encoding the one or more proteins involved in 4CA3O degradation comprises fadD17. In certain embodiments, the modified microbial cell further comprises one or more upstream enzymes that produce androstenedione, androstenedione and / or dihydrogenase with reduced activity. In certain embodiments, the reduced activity refers to a reduction relative to a control cell. In certain embodiments, the one or more upstream enzymes that produce androstenedione, androstenedione and / or dihydrogenase (e.g., androstenedione) include 3-ketosteroid-δ-1-dehydrogenase. In certain embodiments, the one or more upstream enzymes producing androstenedione, androstenedione and / or biredoalcohol are encoded by kstD, chsH3, hsd4A and / or ltp2 genes. In certain embodiments, the modified microbial cells described herein comprise one or more knockouts comprising: (a) chsE3, chsE1, chsE2, casC3, kstD and chsH3; (b) chsE3, chsE1, chsE2, casC3, kstD, chsH3 and chsE5; (c) chsE3, chsE1, chsE2, casC3, kstD, chsH3, chsE4 and fadD17; or (d) chsE3, chsE1, chsE2, casC3, kstD, chsH3, chsE4, chsE5 and fadD17.

[0022] In certain embodiments, the modified microbial cell further comprises one or more proteins involved in the degradation of LCA and / or UDCA with reduced activity. In certain embodiments, the reduced activity refers to a decrease relative to control cells. In certain embodiments, the one or more proteins involved in the degradation of lithocholic acid (LCA) and / or ursodeoxycholic acid (UDCA) participate in the oxidation of 3α-hydroxyl and the oxidation of 5-carbon positions to form a double bond between the 4 and 5 positions of LCA and / or UDCA; or participate in the oxidation, epimerization and dehydroxylation of the 7β hydroxyl of UDCA. In certain embodiments, the reduced activity is achieved by one or more of the following means: gene deletion (e.g., gene knockout), gene disruption, altering the regulation of a gene, replacing a native promoter with a less active promoter, random mutagenesis, genome-wide knockout based on CRISPR, and expression of proteins or protein variants with reduced activity.

[0023] In certain embodiments, the modified microbial cell further comprises one or more enzymes involved in the conversion of 4CA3O to 3-keto-LCA with increased activity. In certain embodiments, the increased activity refers to an increase relative to a control cell. In certain embodiments, the one or more enzymes involved in the conversion of 4CA3O to 3-keto-LCA include 5β-steroid reductase (5βR). In certain embodiments, the 5βR is Tarenaya hassleriana 5βR (e.g., SEQ ID NO: 77) or Raphanus sativus 5βR (e.g., SEQ ID NO: 95). In certain embodiments, the modified microbial cell further comprises one or more enzymes involved in the conversion of 3-keto-LCA to LCA with increased activity, wherein the increase in activity refers to an increase relative to a control cell. In certain embodiments, the one or more enzymes involved in the conversion of 3-keto-LCA to LCA include 3α-hydroxysteroid dehydrogenase (3α-HSD). In certain embodiments, the 3α-HSD is Eggerthella sp. 3α-HSD (e.g., SEQ ID NO: 99) or Ruminococcus gnavus 3α-HSD (e.g., SEQ ID NO: 104). In certain embodiments, the activity of the one or more enzymes involved in the conversion of 4CA3O to 3-keto-LCA or 3-keto-LCA to LCA is increased by expressing an enzyme or enzyme variant having an increased expression level relative to the native enzyme.

[0024] In certain embodiments, the modified microbial cell further comprises one or more enzymes involved in the conversion of LCA to UDCA with increased activity. In certain embodiments, the increased activity refers to an increase relative to a control cell. In certain embodiments, the one or more enzymes involved in the conversion of LCA to UDCA include 7β-hydroxylases and / or one or more redox chaperones (e.g., ferredoxin and ferredoxin reductase, or cytochrome P450 reductase). In certain embodiments, the 7β-hydroxylase is Allokutzneria albata 7β-hydroxylase (e.g., SEQ ID NO: 109). In certain embodiments, the one or more redox chaperones include or are derived from Synechocystis sp. ferredoxin (e.g., SEQ ID NO: 137) and Synechocystis sp. ferredoxin reductase (e.g., SEQ ID NO: 136). In certain embodiments, the activity of the one or more enzymes involved in the conversion of LCA to UDCA is increased by expressing the enzyme or enzyme variant at an increased expression level relative to the native enzyme.

[0025] In certain embodiments, the modified microbial cell described herein comprises (a) a first expression vector comprising, from the 5' end to the 3' end, a first gene sequence encoding 7β-hydroxylase, a second gene sequence encoding ferredoxin, and a third gene sequence encoding ferredoxin reductase, in sequence or in any order; (b) a second expression vector comprising, from the 5' end to the 3' end, a fourth gene sequence encoding 5βR and a fifth gene sequence encoding 3α-HSD, in sequence or in any order. In certain embodiments, the first expression vector and / or the second expression vector further comprises a promoter (e.g., Pg13, Psmo3, or PkshA). In certain embodiments, the first expression vector comprises the Psmo3 promoter, and the second expression vector comprises the PkshA or Pg13 promoter.

[0026] In certain embodiments, the modified microbial cell described herein comprises: (a) a first expression vector comprising, from 5' to 3' end, a Psmo3 promoter, a first gene sequence encoding Allokutzneria albata 7β-hydroxylase, a second gene sequence encoding a Synechocystis sp. ferredoxin, and a third gene sequence encoding a ferredoxin reductase derived from Synechocystis sp. ferredoxin reductase; and a second expression vector comprising, from 5' to 3' end, a PkshA promoter, a fourth gene sequence encoding Tarenaya hassleriana 5βR, and a fifth gene sequence encoding Eggerthella sp. 3α-HSD; (b) a first expression vector comprising, from 5' to 3' end, a Psmo3 promoter, a second gene sequence encoding Allokutzneria albata 7β-hydroxylase, and a third gene sequence encoding a ferredoxin reductase derived from Synechocystis sp. ferredoxin reductase. albata 7β-hydroxylase, a second gene sequence encoding a ferredoxin from Synechocystis sp., and a third gene sequence encoding a ferredoxin reductase derived from Synechocystis sp.; and a second expression vector comprising, from 5' to 3' end, a PkshA promoter, a fourth gene sequence encoding a 5βR from Tarenaya hassleriana, and a fifth gene sequence encoding a 3α-HSD from Ruminococcus gnavus; (c) a first expression vector comprising, from 5' to 3' end, a Psmo3 promoter, a first gene sequence encoding a 7β-hydroxylase from Allokutzneria albata, a second gene sequence encoding a ferredoxin from Synechocystis sp., and a third gene sequence encoding a ferredoxin reductase derived from Synechocystis sp. sp.) ferredoxin reductase; and a second expression vector comprising, from the 5' end to the 3' end, a Pg13 promoter, a fourth gene sequence encoding Tarenaya hassleriana 5βR, and a fifth gene sequence encoding Eggerthella sp. 3α-HSD; (d) a first expression vector comprising, from the 5' end to the 3' end, a Psmo3 promoter, a first gene sequence encoding Allokutzneria albata 7β-hydroxylase, a first gene sequence encoding Synechocystis sp.) a second gene sequence encoding a ferredoxin, and a third gene sequence encoding a ferredoxin reductase derived from Synechocystis sp.; and a second expression vector comprising, from 5' to 3' end, a PkshA promoter, a fourth gene sequence encoding a Raphanus sativus 5βR, and a fifth gene sequence encoding an Eggerthella sp. 3α-HSD; or (e) a first expression vector comprising, from 5' to 3' end, a Psmo3 promoter, a first gene sequence encoding an Allokutzneria albata 7β-hydroxylase, a second gene sequence encoding a ferredoxin, and a fifth gene sequence encoding an Eggerthella sp. sp.) ferredoxin reductase; and a second expression vector comprising, from the 5' end to the 3' end, a Pg13 promoter, a fourth gene sequence encoding Tarenaya hassleriana 5βR, and a fifth gene sequence encoding Ruminococcus gnavus 3α-HSD.

[0027] In one aspect, the present disclosure relates to a method of culturing an engineered microbial cell as described herein, comprising culturing the cell under conditions suitable for producing 4CA3O, 3-keto-LCA, LCA and / or UDCA (e.g., in the presence of a phytosterol (e.g., β-sitosterol, β-sitostanol, campesterol, stigmasterol and / or brassicasterol)), optionally wherein the method further comprises recovering 4CA3O, 3-keto-LCA, LCA and / or UDCA from the culture.

[0028] In certain embodiments, the modified microbial cell is a bacterial cell (e.g., a Mycobacterium species). In certain embodiments, the bacterial cell is a Mycobacterium neoaurum cell (e.g., Mycobacterium neoaurum NRRL B-3805).

[0029] In one aspect, the present disclosure relates to a culture of modified microbial cells comprising the modified microbial cells described herein. In certain embodiments, the culture comprises an amount greater than 0.01 mg / L, 0.05 mg / L, 0.1 mg / L, 0.5 mg / L, 1 mg / L, 5 mg / L, 10 mg / L, 50 mg / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, 55 g / L, 60 g / L, 65 g / L, 70 g / L, 75 g / L, 80 g / L, 85 g / L, 90 g / L, 95 g / L or 100 g / L of progesterone, 4CA3O, 3-keto-LCA, LCA and / or UDCA.

[0030] In one aspect, the present disclosure relates to a method for producing pregnenolone and / or progesterone, comprising contacting the engineered microbial cells described herein or a cell extract thereof with a sterol (e.g., a phytosterol), thereby producing pregnenolone and / or progesterone.

[0031] In one aspect, the present disclosure relates to a method for producing pregnenolone and / or progesterone, comprising contacting a side chain cleavage P450 enzyme with a sterol (e.g., a phytosterol) to produce pregnenolone and / or progesterone. In certain embodiments, the side chain cleavage P450 enzyme is any one of the side chain cleavage P450 enzymes in Table 1, or a derivative thereof. In certain embodiments, the methods described herein further comprise contacting a ferredoxin and / or a ferredoxin reductase with a side chain cleavage P450 enzyme and a sterol (e.g., a phytosterol). In certain embodiments, the ferredoxin is any one of the ferredoxin or a derivative thereof in Table 2, and in certain embodiments, the ferredoxin reductase is any one of the ferredoxin reductases in Table 3, or a derivative thereof.

[0032] In one aspect, the present disclosure relates to a method for producing 3-keto-lithocholic acid, comprising contacting the engineered microbial cell described herein or a cell extract thereof with 4-cholic acid-3-one, thereby producing 3-keto-lithocholic acid.

[0033] In one aspect, the present disclosure relates to a method for producing 3-keto-lithocholic acid, comprising contacting a 5β-steroid reductase with 4-cholic acid-3-one, thereby producing 3-keto-lithocholic acid. In certain embodiments, the 5β-steroid reductase is any one of the 5β-steroid reductases listed in Table 4 or a derivative thereof.

[0034] In one aspect, the present disclosure relates to a method for producing lithocholic acid, comprising contacting an engineered microbial cell described herein or a cell extract thereof with 3-keto-lithocholic acid, thereby producing lithocholic acid.

[0035] In one aspect, the present disclosure relates to a method for producing lithocholic acid, comprising contacting a 3α-hydroxysteroid dehydrogenase with 3-keto-lithocholic acid, thereby producing lithocholic acid. In certain embodiments, the 3α-hydroxysteroid dehydrogenase is any one of the 3α-hydroxysteroid dehydrogenases listed in Table 5 or a derivative thereof.

[0036] In one aspect, the present disclosure relates to a method for producing lithocholic acid, comprising contacting the engineered microbial cell described herein or a cell extract thereof with 4-cholic acid-3-one, thereby producing lithocholic acid.

[0037] In one aspect, the present disclosure relates to a method for producing lithocholic acid, the method comprising contacting 4-cholic acid-3-one with a 5β-steroid reductase and a 3α-hydroxysteroid dehydrogenase, thereby producing lithocholic acid. In certain embodiments, the 5β-steroid reductase is any one of the 5β-steroid reductases listed in Table 4 or a derivative thereof, and in certain embodiments, the 3α-hydroxysteroid dehydrogenase is any one of the 3α-hydroxysteroid dehydrogenases listed in Table 5 or a derivative thereof.

[0038] In one aspect, the present disclosure relates to a method for producing ursodeoxycholic acid, comprising contacting the engineered microbial cells described herein or a cell extract thereof with lithocholic acid, thereby producing ursodeoxycholic acid.

[0039] In one aspect, the present disclosure relates to a method for producing ursodeoxycholic acid, comprising contacting a 7β-hydroxylase with lithocholic acid, thereby producing ursodeoxycholic acid. In certain embodiments, the 7β-hydroxylase is any one of the 7β-hydroxylases listed in Table 6 or a derivative thereof. In certain embodiments, the methods described herein further comprise contacting a ferredoxin and / or a ferredoxin reductase with the 7β-hydroxylase and lithocholic acid. In certain embodiments, the ferredoxin is any one of the ferredoxin or a derivative thereof listed in Table 2, and in certain embodiments, the ferredoxin reductase is any one of the ferredoxin reductases listed in Table 3 or a derivative thereof.

[0040] In one aspect, the present disclosure relates to a method for producing ursodeoxycholic acid, comprising contacting the engineered microbial cell described herein or a cell extract thereof with 3-keto-lithocholic acid, thereby producing ursodeoxycholic acid.

[0041] In one aspect, the present disclosure relates to a method for producing ursodeoxycholic acid, the method comprising: contacting a 3α-hydroxysteroid dehydrogenase and a 7β-hydroxylase with 3-keto-lithocholic acid, thereby producing ursodeoxycholic acid. In certain embodiments, the 3α-hydroxysteroid dehydrogenase is any one of the 3α-hydroxysteroid dehydrogenases or derivatives thereof in Table 5, and in certain embodiments, the 7β-hydroxylase is any one of the 7β-hydroxylase or derivatives thereof in Table 6. In certain embodiments, the methods described herein further comprise: contacting a ferredoxin and / or a ferredoxin reductase with the 3α-hydroxysteroid dehydrogenase, the 7β-hydroxylase, and the 3-keto-lithocholic acid. In certain embodiments, the ferredoxin is any one of the ferredoxin or derivatives thereof in Table 2, and in certain embodiments, the ferredoxin reductase is any one of the ferredoxin reductases or derivatives thereof in Table 3.

[0042] In one aspect, the present disclosure relates to a method for producing ursodeoxycholic acid, comprising contacting the engineered microbial cell described herein or a cell extract thereof with 4-cholic acid-3-one, thereby producing ursodeoxycholic acid.

[0043] In one aspect, the present disclosure relates to a method for producing ursodeoxycholic acid, comprising contacting a 5β-steroid reductase, a 3α-hydroxysteroid dehydrogenase, and a 7β-hydroxylase with 4-cholic acid-3-one, thereby producing ursodeoxycholic acid. In certain embodiments, the 5β-steroid reductase is any one of the 5β-steroid reductases listed in Table 4, or a derivative thereof, in certain embodiments, the 3α-hydroxysteroid dehydrogenase is any one of the 3α-hydroxysteroid dehydrogenases listed in Table 5, or a derivative thereof, and in certain embodiments, the 7β-hydroxylase is any one of the 7β-hydroxylases listed in Table 6, or a derivative thereof. In certain embodiments, the methods described herein further comprise contacting a ferredoxin and / or ferredoxin reductase with the 5β-steroid reductase, the 3α-hydroxysteroid dehydrogenase, the 7β-hydroxylase, and 4-cholic acid-3-one. In certain embodiments, the ferredoxin is any one of the ferredoxin proteins listed in Table 2 or a derivative thereof, and in certain embodiments, the ferredoxin reductase is any one of the ferredoxin reductases listed in Table 3 or a derivative thereof.

[0044] As used herein, unless otherwise indicated, the enzymes in the present disclosure (e.g., side-chain cleavage P450 enzymes, ferredoxins, ferredoxin reductases, dehydrogenases, etc.) include wild-type enzymes and variants thereof. Variants may have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the wild-type enzyme, but the variants retain similar function or activity, or may even have improved function or activity. For example, the "side-chain cleavage P450 enzyme" catalyzes the conversion of cholesterol to pregnenolone. The term "side-chain cleavage P450 enzyme" includes wild-type side-chain cleavage P450 enzymes and variants thereof, wherein the variants have substantially similar or even better function or activity. Similarly, "ferredoxin" includes wild-type ferredoxin and variants thereof, wherein the variants have substantially similar or even better function or activity. "Feredoxin reductase" includes wild-type ferredoxin reductase and variants thereof, wherein the variant has substantially similar or better function or activity. Likewise, Novosphingobium sp. PP1Y side-chain cleavage P450 enzyme, Novosphingobium subterraneum side-chain cleavage P450 enzyme, Synechococcus elongatus ferredoxin, Synechococcus elongatus ferredoxin reductase, Chlamydomonas reinhardtii ferredoxin, Spinacia oleracea ferredoxin, Spinacia oleracea ferredoxin reductase, cholesterol oxidase, hydroxysteroid dehydrogenase, ketosteroid isomerase, thioesterase, 3α-hydroxysteroid dehydrogenase (3α-HSD), 5β-steroid reductase (5βR), 7β-hydroxylase, cytochrome P450 reductase, and the like include wild-type enzymes and their respective variants.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of any conflict, the present specification (including definitions) will prevail.

[0046] Other features and advantages of the invention will be set forth in the following detailed description and drawings, and in the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a schematic diagram showing the two metabolic pathways for converting phytosterols (specifically β-sitosterol) to progesterone. The upper pathway proceeds via β-sitosterol, while the lower pathway proceeds via pregnenolone. P450: cytochrome P450; RPP: redox chaperone protein; cho: cholesterol oxidase; hsdD: hydroxysteroid dehydrogenase; ksi: ketosteroid isomerase.

[0048] Figure 2 This is a graph of the LC-MS peak area for progesterone in a 96-well plate assay. This assay tested Mycobacterium neoaurum NRRL B-3805 transformed with different replication plasmids containing a side-chain cleavage P450 gene paired with ferredoxin and ferredoxin reductase. Cultures of the strains were incubated with 10 g / L of 70% β-sitosterol for three days. At least two replicates were tested for each strain. P001 is an empty plasmid control.

[0049] Figure 3 This is a graph of the RapidFire peak area for androstenedione in a 96-well plate assay. This assay tested different strains with different combinations of gene knockouts. Strain cultures were incubated with 10 g / L of 70% β-sitosterol for three days. Each strain was tested in at least two replicates.

[0050] Figure 4 Figure 2 is a graph of the GC peak areas of progesterone and androstenedione (AD) in a 96-well plate assay. This assay tested wild-type (WT) strains (NRRL B-3805) or smo3 knockout mutants (KO_smo3) transformed with various P450 replication plasmids. Strain cultures were incubated with 10 g / L of 70% β-sitosterol for three days. Each strain was tested in at least two replicates. P001 is an empty plasmid control.

[0051] Figure 5 This graph shows the GC peak area for progesterone in a 96-well plate assay. This assay tested a smo3 knockout mutant strain (KO_smo3) transformed with replication plasmids containing different side-chain cleavage cytochrome P450 genes. Cultures were incubated with 10 g / L 70% β-sitosterol for three days. Each strain was tested in at least two replicates.

[0052] Figure 6This graph shows the GC peak area for progesterone in a 96-well plate assay. This assay tested a smo3 knockout mutant strain (KO_smo3) transformed with replication plasmids expressing the P450 gene CYP_9SPHN_2 and different combinations of redox chaperone proteins. Cultures of the strain were incubated with 10 g / L of 70% β-sitosterol for three days. Each strain was tested in at least two replicates.

[0053] Figure 7 This is a graph of the progesterone RapidFire peak area from a 96-well plate assay. This assay tested mutant strains with multiple gene knockouts transformed with replicative plasmids expressing different fusion protein designs. Strain cultures were incubated with 10 g / L of 70% β-sitosterol for three days. Each strain was tested in at least two replicates. The CYP_XXXXX_1>FDX_SPIOL_1>FDXR_SPIOL_1 plasmid design contains an operon in which cytochrome P450, ferredoxin, and ferredoxin reductase are expressed as separate enzyme components. All other plasmid designs contain genes encoding fusion proteins of two of these three components.

[0054] Figure 8A Schematic diagram showing the metabolic pathway for the conversion of phytosterols, particularly β-sitosterol, to 4-cholic acid-3-one (4CA3O), 3-keto-lithocholic acid (3-keto-LCA), lithocholic acid (LCA), and ursodeoxycholic acid (UDCA).

[0055] Figure 8B is a schematic diagram showing the metabolic pathway for the conversion of phytosterols, particularly β-sitosterol, to androstenedione (AD) and androstenedione (ADD).

[0056] Figure 9 Figure 2 is a graph of the LC-MS peak area of ​​4-cholic acid-3-one (4CA3O) and the GC peak area of ​​androstenedione (AD) in a 96-well plate assay. The assay used cultures of several mutant Mycobacterium strains incubated with 10 g / L of 70% β-sitosterol for one and three days. Each strain was tested in at least four replicates.

[0057] Figure 10 The amino acid sequences discussed in this disclosure are listed.

[0058] Figure 11 The DNA sequences of the genes discussed in this disclosure are listed.

[0059] Figure 12Figure 2 is a plot of the LC-UV peak areas of 4-cholic acid-3-one and 4-androstenedione (AD) in a 96-well plate assay. The assay used cultures of several mutant Mycobacterium strains incubated with 10 g / L of 70% β-sitosterol for three days. Each strain was tested in at least four replicates.

[0060] Figure 13 This graph shows the LC-MS peak areas of UDCA and 4-androstenedione (AD) in a 96-well plate assay. This assay tests the ability of several mutant Mycobacterium strains to degrade UDCA. Strain cultures were incubated with 500 mg / L UDCA for three days. At least six replicates were performed for each strain. A cell-free sample served as an experimental control.

[0061] Figure 14 Figure 2 is a plot of the LC-MS peak area for 3-keto-lithocholic acid (3-keto-LCA) in a 96-well plate assay. This assay tested several mutant Mycobacterium strains expressing different candidate 5-β-reductases. Strain cultures were incubated with 10 g / L of 70% β-sitosterol. Each strain was tested in at least three replicates. A negative control sample was the parent strain harboring an empty replicating plasmid.

[0062] Figure 15 Figure 2 is a plot of the LC-MS peak area for lithocholic acid (LCA) in a 96-well plate assay. This assay tested several mutant Mycobacterium strains expressing different 5β-reductases, 5BR_ARATH_1, and candidate 3α-hydroxysteroid dehydrogenases. Strain cultures were incubated with 10 g / L of 70% β-sitosterol. Each strain was tested in at least six replicates. A negative control sample was the parent strain harboring an empty replicating plasmid.

[0063] Figure 16 Figure 2 is a plot of the LC-MS peak area for ursodeoxycholic acid (UDCA) in a 96-well plate assay. This assay tested several mutant Mycobacterium strains expressing different candidate 7β-hydroxylases and redox chaperones: ferredoxin (FDX_SYNY4_2) and ferredoxin reductase (FDXR_SYNY4_2). Cultures of the strains were incubated with 1 g / L lithocholic acid. Each strain was tested in at least three replicates. Negative controls included the parent strain harboring an empty replicating plasmid.

[0064] Figure 17This figure shows the LC-MS peak areas for ursodeoxycholic acid (UDCA) and lithocholic acid (LCA) from a 96-well plate assay. This assay tested several mutant Mycobacterium strains, each containing replicating plasmids that co-express the enzymes required for UDCA production from phytosterols. These included a candidate 7β-hydroxylase, a cytochrome P450 redox chaperone, a 5β-reductase, and a 3α-hydroxysteroid dehydrogenase. Cultures of the strains were incubated with 10 g / L of 70% β-sitosterol. Each strain was tested in at least three replicates. P009 is a negative control plasmid that does not express any enzymes in this pathway. DETAILED DESCRIPTION

[0065] The present disclosure describes a method for producing small molecule compounds progesterone and / or ursodeoxycholic acid (UDCA) by fermentation with a microbial host. Non-native enzymes or proteins can be introduced into suitable microbial hosts for industrial fermentation of large-scale chemical products. For example, expressing non-native enzymes, such as side chain cleavage P450 enzymes and redox chaperones, in a microbial host can increase the production of progesterone; expressing non-native enzymes, such as 5β-steroid reductase (5βR), 3α-hydroxysteroid dehydrogenase (3α-HSD), 7β-hydroxylase and / or one or more redox chaperones in a microbial host can increase the production of UDCA and one or more intermediates in the UDCA pathway (e.g., 4-cholic acid-3-one (4CA3O), 3-keto-lithocholic acid (3-keto-LCA) and / or lithocholic acid (LCA)). Certain native genes encoding enzymes involved in the production of unwanted byproducts (e.g., androstenedione) and / or the degradation of precursor molecules (e.g., phytosterols, 4CA3O, 3-keto-LCA and / or LCA) can also be disrupted (e.g., knocked out) to further increase progesterone or UDCA production.

[0066] In certain embodiments, the present disclosure describes methods for producing intermediates in the UDCA pathway (e.g., 4CA3O, 3-keto-LCA, and / or LCA). In certain embodiments, such compounds can be isolated from fermentations of microbial hosts (e.g., Mycobacterium) and converted to UDCA by additional chemical and / or biological processes.

[0067] The term "fermentation" is used herein to refer to a process by which microbial cells convert one or more substrates into a desired product (eg, progesterone and / or ursodeoxycholic acid) through one or more bioconversion steps, without any chemical conversion steps.

[0068] The term "variant" refers to a molecule that has certain differences in its base or amino acid sequence compared to a reference polynucleotide or polypeptide (naturally occurring), respectively, and these differences are artificially synthesized or naturally occurring. These differences include substitutions, insertions, deletions, or any desired combination of such changes in the natural polynucleotide encoding the amino acid sequence.

[0069] The term "engineered" when referring to a cell is used herein to indicate that the cell contains at least one artificially introduced targeted genetic alteration that distinguishes the engineered cell from a naturally occurring cell.

[0070] The term "native" is used herein to refer to a cellular component, such as a polynucleotide or polypeptide, that is naturally present in a particular cell. A native polynucleotide or polypeptide is endogenous to the cell.

[0071] The term "non-native state" when referring to a polynucleotide or polypeptide is used herein to refer to a polynucleotide or polypeptide that does not naturally occur in a particular cell.

[0072] When referring to the environment of gene expression, the term "non-native" refers to the expression of a gene in any context other than the genomic and cellular context in which it is naturally expressed. A gene expressed in a non-native manner may have the same nucleotide sequence as the corresponding gene in the host cell but may be expressed from a vector or at an integration point in the genome that is different from the native gene locus.

[0073] The term "heterologous" is used herein to describe a polynucleotide or polypeptide introduced into a host cell. The term encompasses polynucleotides or polypeptides derived from organisms, species or bacterial strains that are different from the host cell. In this case, heterologous polynucleotides or polypeptides have a sequence different from any sequence found in the same host cell. However, the term also encompasses polynucleotides or polypeptides with a sequence identical to the sequence found in the host cell, wherein the polynucleotides or polypeptides are present in a background different from the native sequence (for example, heterologous polynucleotides can be connected to different promoters and inserted into a genomic location different from the native sequence). Therefore, "heterologous expression" encompasses the expression of the non-native sequence of the host cell, and the native expression of the native sequence of the host cell under non-native state background.

[0074] The term "wild-type" when referring to a polynucleotide or polypeptide refers to any polynucleotide having a nucleotide sequence, or polypeptide having an amino acid sequence, found in a naturally occurring organism, regardless of the source of the molecule; that is, the term "wild-type" refers to sequence characteristics regardless of whether the molecule is purified from a natural source, purified after recombinant expression, or synthesized. The term "wild-type" is also used to refer to naturally occurring cells.

[0075] A "control cell" is a cell that is otherwise identical to the engineered cell being tested, including cells of the same species as the engineered cell, but lacking the particular genetic modification being tested in the engineered cell.

[0076] Enzymes are identified herein by the reactions they catalyze, and unless otherwise stated, enzymes refer to any polypeptide capable of catalyzing the reactions identified. Unless otherwise stated, enzymes can be derived from any organism and can have natural or mutant amino acid sequences. As is well known, enzymes may have multiple functions and / or multiple names sometimes due to the differences in the organisms from which they are derived. Enzyme names used herein encompass orthologs, including enzymes that may have one or more additional functions or different names.

[0077] The term "progesterone" refers to the 21 H 30 The term "ursodeoxycholic acid" or "UDCA" refers to a chemical compound with the formula C 24 H 40 The term "4-cholic acid-3-one" or "4CA3O" refers to a chemical compound with the formula C 24 H 36 The term "3-keto-lithocholic acid" or "3-keto-LCA" refers to a chemical compound with the formula C 24 H 38 O3 chemical compound (CAS#: 1553-56-6). The term "lithocholic acid" or "LCA" refers to a compound with the molecular formula C 24 H 40 Chemical compound of O3 (CAS#: 434-13-9).

[0078] The term "sequence identity" refers to two or more sequences that are identical or have a specified percentage of amino acid residues or nucleotides that are identical when compared and aligned for maximum correspondence as measured using a sequence comparison algorithm or by visual inspection.

[0079] In order to perform sequence comparisons to determine percent nucleotide or amino acid sequence identity, one sequence is typically compared to a "reference sequence" as a "test" sequence. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, and if necessary, subsequence coordinates are specified, along with sequence algorithm program parameters. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters. Sequence alignments for comparison can be performed using BLAST set to default parameters.

[0080] As used herein, the term "titer" refers to the mass of product (eg, progesterone, 4CA3O, 3-keto-LCA, LCA, and / or UDCA) produced in a microbial cell culture divided by the culture volume.

[0081] As used herein, with respect to recovering progesterone, 4CA3O, 3-keto-LCA, LCA and / or UDCA from a cell culture, "recovering" means separating the progesterone, 4CA3O, 3-keto-LCA, LCA and / or UDCA from at least one other component of the cell culture medium.

[0082] In certain embodiments, the phytosterols described herein include phytosterols and phytostanols. In certain embodiments, phytosterols of different compositions can be used as substrates for the conversion of progesterone, 4CA3O, 3-keto-LCA, LCA and / or UDCA. In certain embodiments, the phytosterol composition is 36-80% β-sitosterol, 6-34% β-sitostanol, 4-25% campesterol, and optionally has 0-14% campesterol, <1% stigmasterol, <1% brassicasterol, 0-2% other minor sterols. In certain embodiments, the phytosterol composition is 43-52% β-sitosterol, 0-3% β-sitostanol, 24-28% campesterol, 14-24% stigmasterol, and optionally has 0-2% campesterol, 0-9% brassicasterol, <3% other minor sterols. In certain embodiments, the phytosterols described herein comprise one or more (eg, 1, 2, 3, 4, or 5) β-sitosterol, β-sitostanol, campesterol, stigmasterol, and brassicasterol.

[0083] In certain embodiments, the phytosterols or phytosterol compositions described herein are used as substrates for the production of any of the compounds described herein (e.g., pregnenolone, progesterone, 4CA3O, 3-keto-LCA, LCA, and / or UDCA).

[0084] In certain embodiments, production of a compound described herein (e.g., progesterone or UDCA) or an intermediate thereof (e.g., pregnenolone, 4CA3O, 3-keto-LCA, or LCA) is performed in vivo, for example, by a bioconversion process in which a substrate is provided to an engineered microbial cell (such as any engineered microbial cell described herein) to produce the compound or intermediate thereof. In certain embodiments, production of a compound described herein (e.g., progesterone or UDCA) or an intermediate thereof (e.g., pregnenolone, 4CA3O, 3-keto-LCA, or LCA) is performed in vitro, e.g., by a biocatalytic process whereby a substrate is provided to one or more purified or partially purified enzymes (e.g., any side-chain cleavage P450 enzyme described herein, any ferredoxin described herein, any ferredoxin reductase described herein, any 5β-steroid reductase described herein, any 3α-hydroxysteroid dehydrogenase described herein, any 7β-hydroxylase described herein, or any combination thereof) or cell extracts (e.g., cell extracts of any engineered microbial cells described herein) to produce the compound.

[0085] Engineering microbes to produce progesterone

[0086] Progesterone biosynthesis pathway

[0087] Progesterone is a steroid produced naturally in the human body and is also an active pharmaceutical ingredient (API) that is commonly used in female hormone replacement therapy. Most current production methods use chemical methods that require harsh chemicals and solvents. The present disclosure proposes a method for producing progesterone directly from plant sterols or alternative sterols (such as cholesterol) by fermentation, which avoids any chemical steps. The full biosynthesis of progesterone using simple carbon sources is still in the development stage, which requires the modification of many enzymatic steps and the regulation of natural pathways. The method disclosed herein uses plant sterols as starting materials, which are already structurally similar to the final product. Therefore, the conversion only requires two enzymatic steps, which greatly simplifies the development of strains.

[0088] Progesterone is used as part of hormone replacement therapy for women who have gone through menopause (a turning point in life) and have not yet had a hysterectomy (surgery to remove the uterus). Hormone replacement therapy typically includes estrogen, which is used to treat menopausal symptoms and reduce the risk of certain medical conditions. However, estrogen can also cause abnormal thickening of the lining of the uterus, increasing the risk of uterine cancer. Progesterone helps prevent this thickening and reduces the risk of uterine cancer. Progesterone is also used to restore menstruation in women of childbearing age who had regular periods but later stopped. Progesterone is a member of a class of medications called progestogens (female hormones). As part of hormone replacement therapy, progesterone works by reducing the amount of estrogen in the uterus. Progesterone restores menstruation by replacing the natural progesterone that some women are missing.

[0089] Figure 1 Two possible biosynthetic pathways for the conversion of phytosterols to progesterone are shown. In the first pathway described, the initial step is the conversion of phytosterols to phytosterone, for example, the conversion of β-sitosterol to β-sitosterone. Other phytosterols, such as campesterol and other sterols, such as cholesterol, can also be used as substrates. This initial step involves the oxidation of the 3-hydroxyl group to a ketone and the isomerization of the 5,6-double bond to the 4,5-position. This step is catalyzed by several known enzymes, including cholesterol oxidase (CHO), hydroxysteroid dehydrogenase (HSDD), and / or ketosteroid isomerase (KSI). In the second step of the first pathway, the side chain of the phytosterone is cleaved by cytochrome P450 to produce progesterone. The cytochrome P450 is activated by redox chaperones (e.g., ferredoxin and ferredoxin reductase). In the second pathway, the order of steps is reversed. First, the side chain of the phytosterol is cleaved to form pregnenolone. Subsequently, pregnenolone is converted to progesterone by oxidation of the 3-hydroxyl group and isomerization of the double bond.

[0090] In certain embodiments, the engineered microbial cell further expresses heterologous or additional copies of native cholesterol oxidase (cho), hydroxysteroid dehydrogenase (hsdD), and / or ketosteroid isomerase (ksi).

[0091] Modification of microbial progesterone production

[0092] Generally, genes encoding enzymes can be introduced and expressed into microbial cells by conventional genetic modification techniques, thereby making the modified microbial cells have any active side chain cleavage P450 enzymes and redox chaperone proteins (e.g., ferredoxin and ferredoxin reductase). Suitable side chain cleavage P450 enzymes and redox chaperone proteins (e.g., ferredoxin and ferredoxin reductase) can be derived from any source, including plants, archaea, fungi, gram-positive bacteria and gram-negative bacteria.

[0093] One or more copies of any of these genes can be introduced into selected microbial host cells. If more than one gene copy is introduced, these copies can have identical or different nucleotide sequences. In certain embodiments, one or two (or all) heterologous genes are expressed by a strong constitutive promoter (e.g., Ptac or Ppms). In certain embodiments, heterologous genes are expressed by an inducible promoter. Heterologous genes can be selected to be codon optimized to enhance expression in selected microbial host cells.

[0094] In certain embodiments, the gene ID, source, and amino acid sequence of the side chain cleavage P450 enzymes described herein are listed in the table below. For example, MBS0474250.1 is the GenBank ID of CYP_9SPHN_7 in the National Center for Biotechnology Information (NCBI) database.

[0095] Table 1 Side chain cleavage P450 enzymes used in this disclosure

[0096]

[0097]

[0098] In certain embodiments, the gene ID, source, and amino acid sequence of the ferredoxins described herein are listed in the table below.

[0099] Table 2. Ferredoxins used in this disclosure

[0100]

[0101] In certain embodiments, the gene ID, source, and amino acid sequence of the ferredoxin reductases described herein are listed in the table below.

[0102] Table 3. Ferredoxin reductases used in this disclosure

[0103]

[0104]

[0105] In certain embodiments, the modified microbial cells described herein express a non-native side chain cleavage P450 enzyme (e.g., any one of the side chain cleavage P450 enzymes in Table 1). In certain embodiments, the modified microbial cells described herein express one or more non-native redox chaperone proteins, such as a non-native ferredoxin, a non-native ferredoxin reductase, or both. In certain embodiments, the non-native ferredoxin is any one of the ferredoxins in Table 2. In certain embodiments, the non-native ferredoxin reductase is any one of the ferredoxin reductases in Table 3. In certain embodiments, the side chain cleavage P450 enzyme is capable of increasing the production of progesterone by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or by at least 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, 50-fold, 51-fold, 52-fold, 53-fold, 54-fold, 55-fold, 56-fold, 57-fold, 58-fold 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times or 1000 times. In some embodiments, the non-native ferredoxin and / or non-native ferredoxin reductase can increase the production of progesterone by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or at least 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, 50-fold, 51-fold, 52-fold, 53-fold, 54-fold, 55-fold, 56-fold, 57-fold, 58-fold, 59-fold, 60- 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times or 1000 times.

[0106] In one aspect, the modified microbial cells described herein contain vectors suitable for expressing proteins (e.g., any combination of side chain cleavage P450 enzymes and redox chaperone proteins) in host microbial cells. In certain embodiments, the vectors include one or more gene sequences encoding proteins described herein. The gene sequences can be from any source gene or source organism listed in Tables 1-3.

[0107] In certain embodiments, the vectors described herein comprise, from the 5' end to the 3' end, a promoter (e.g., Ptac or Ppms), a first gene sequence encoding any side-chain cleavage P450 enzyme listed in Table 1, a second gene sequence encoding any ferredoxin listed in Table 2, and a third gene sequence encoding any ferredoxin reductase listed in Table 3. In certain embodiments, there is a ribosome binding site upstream of the first gene sequence, the second gene sequence, and the third gene sequence.

[0108] In certain embodiments, the vector comprises a Ppms promoter, a first gene sequence encoding a Novosphingobium subterraneum side-chain cleavage P450 enzyme (e.g., SEQ ID NO: 1 or SEQ ID NO: 2), a second gene sequence encoding a Spinacia oleracea ferredoxin (SEQ ID NO: 5), and a third gene sequence encoding a Spinacia oleracea ferredoxin reductase (e.g., SEQ ID NO: 6). In certain embodiments, the vector comprises a Ppms promoter, a first gene sequence encoding a Novosphingobium aromaticivorans side-chain cleavage P450 enzyme (e.g., SEQ ID NO: 3), a second gene sequence encoding a Spinacia oleracea ferredoxin (SEQ ID NO: 5), and a third gene sequence encoding a Spinacia oleracea ferredoxin reductase (e.g., SEQ ID NO: 6). In certain embodiments, the vector comprises a Ptac promoter, a first gene sequence encoding a Novosphingobium subterraneum side-chain cleavage P450 enzyme (e.g., SEQ ID NO: 1 or SEQ ID NO: 2), a second gene sequence encoding a Spinacia oleracea ferredoxin (SEQ ID NO: 5), and a third gene sequence encoding a Spinacia oleracea ferredoxin reductase (e.g., SEQ ID NO: 6). In certain embodiments, the vector comprises a Ptac promoter, a first gene sequence encoding a Novosphingobium aromaticivorans side-chain cleavage P450 enzyme (e.g., SEQ ID NO: 4), a second gene sequence encoding a Spinacia oleracea ferredoxin (SEQ ID NO: 5), and a third gene sequence encoding a Spinacia oleracea ferredoxin reductase (e.g., SEQ ID NO: 6). In certain embodiments, the vector includes any combination of promoters and proteins shown in Table 7.In certain embodiments, the vector includes a Ppms promoter, a first gene sequence encoding a Novosphingobium sp. side-chain cleavage P450 enzyme (e.g., SEQ ID NO: 18), a second gene sequence encoding a Spinacia oleracea ferredoxin reductase (SEQ ID NO: 5), and a third gene sequence encoding a Spinacia oleracea ferredoxin reductase (e.g., SEQ ID NO: 6).

[0109] In certain embodiments, any two of the first gene sequence, the second gene sequence, and the third gene sequence are linked to express a fusion protein. In certain embodiments, all three of the first gene sequence, the second gene sequence, and the third gene sequence are linked to express a fusion protein. In certain embodiments, any one of the side-chain cleavage P450 enzymes in Table 1 can be fused to any one of the ferredoxin proteins in Table 2. In certain embodiments, any one of the side-chain cleavage P450 enzymes in Table 1 can be fused to any one of the ferredoxin reductases in Table 3. In certain embodiments, any one of the ferredoxin proteins in Table 2 can be fused to any one of the ferredoxin reductases in Table 3. In certain embodiments, the fusion proteins described herein include a linker peptide sequence connecting the two fused proteins. In certain embodiments, the linker peptide sequence is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 54 or 55. In certain embodiments, the fusion protein described herein has an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 47-53. In certain embodiments, the connecting peptide described herein is a rigid connecting peptide. In certain embodiments, the connecting peptide is a flexible connecting peptide.

[0110] Successful construction of a recombinant fusion protein typically requires two elements: component proteins and a linker peptide. The selection of component proteins is based on the desired function of the fusion protein product and is relatively simple in most cases. However, selecting an appropriate linker peptide to connect the protein domains together can be complex and is often overlooked in the design of fusion proteins. Direct fusion of functional domains without a linker peptide can lead to many adverse consequences, including misfolding of the fusion protein, lower protein yield, or impaired biological activity. Therefore, selecting or rationally designing a linker peptide to connect the fusion protein domains is an important but underexplored area in recombinant fusion protein technology. Details of linker peptide design, especially the choice between flexible linker peptides and rigid linker peptides, can be found in, for example, the article by Chen, X. et al. ("Fusion protein linkers: property, design and functionality", Advanced Drug Delivery Reviews 65.10 (2013): 1357-1369), the entire contents of which are incorporated herein by reference.

[0111] In certain embodiments, the vectors described herein comprise, from the 5' end to the 3' end, a Ppms promoter, a first sequence encoding a Novosphingobium sp. side-chain cleavage P450 enzyme (e.g., SEQ ID NO: 18), and a second sequence encoding a fusion protein comprising, from the N-terminus to the C-terminus, a Spinacia oleracea ferredoxin (e.g., SEQ ID NO: 5) and a Spinacia oleracea ferredoxin reductase (e.g., SEQ ID NO: 6). In certain embodiments, the fusion protein has an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 47 or 48.

[0112] In certain embodiments, the vectors described herein comprise, from the 5' end to the 3' end, a Ppms promoter, a first sequence encoding a Novosphingobium sp. side-chain cleavage P450 enzyme (e.g., SEQ ID NO: 18), and a second sequence encoding a fusion protein comprising, from the N-terminus to the C-terminus, a Spinacia oleracea ferredoxin reductase (e.g., SEQ ID NO: 6) and a Spinacia oleracea ferredoxin (e.g., SEQ ID NO: 5). In certain embodiments, the fusion protein has an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 49.

[0113] In certain embodiments, the vectors described herein comprise, from the 5' end to the 3' end, a Ppms promoter, a first sequence encoding a fusion protein, and a second sequence encoding a Spinacia oleracea ferredoxin reductase (e.g., SEQ ID NO: 6), the fusion protein comprising, from the N-terminus to the C-terminus, a Spinacia oleracea ferredoxin (e.g., SEQ ID NO: 5) and a Novosphingobium sp. side-chain cleavage P450 enzyme (e.g., SEQ ID NO: 18). In certain embodiments, the fusion protein has an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 50 or 53.

[0114] In certain embodiments, the vectors described herein comprise, from the 5' end to the 3' end, a Ppms promoter, a first sequence encoding a fusion protein, and a second sequence encoding a Spinacia oleracea ferredoxin reductase (e.g., SEQ ID NO: 6), the fusion protein comprising, from the N-terminus to the C-terminus, a Novosphingobium sp. side-chain cleavage P450 enzyme (e.g., SEQ ID NO: 18) and a Spinacia oleracea ferredoxin (e.g., SEQ ID NO: 5). In certain embodiments, the fusion protein has an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 51 or 52.

[0115] In certain embodiments, expression of the fusion protein can increase progesterone production by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or by at least 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, or more compared to expressing each protein separately using the same vector. , 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times or 1000 times.

[0116] In various embodiments, the progesterone titer is increased in the range of 10-fold to 1000-fold, 20-fold to 500-fold, 50-fold to 400-fold, 10-fold to 300-fold, or any range bounded by any of the foregoing values. These increases are determined relative to the progesterone titer observed in a progesterone-producing microbial cell that does not include the genetic alterations discussed herein. The reference cell may (but does not necessarily) have other genetic alterations intended to increase progesterone production.

[0117] In various embodiments, the progesterone titer achieved by expressing a non-native side-chain cleavage P450 enzyme and one or more non-native redox chaperone proteins is at least 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, or 900 mg / L, or at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 g / L. In various embodiments, the titer range is 10 mg / L to 200 g / L, 100 mg / L to 200 g / L, 1 g / L to 200 g / L, 1 g / L to 150 g / L, 1 g / L to 100 g / L, 1 g / L to 70 g / L, 1 g / L to 50 g / L, 5 g / L to 200 g / L, 5 g / L to 150 g / L, 5 g / L to 100 g / L, 5 g / L to 70 g / L, 5 g / L to 50 g / L, g / L to 50 g / L, 10 g / L to 200 g / L, 10 g / L to 150 g / L, 10 g / L to 100 g / L, 10 g / L to 70 g / L, 10 g / L to 50 g / L, 20 g / L to 200 g / L, 20 g / L to 150 g / L, 20 g / L to 100 g / L, 20 g / L to 70 g / L, 20 g / L to 50 g / L, or any range defined by any of the above values.

[0118] Reduced consumption of progesterone and / or its precursors

[0119] Methods for increasing progesterone production and reducing unwanted byproducts in microbial cells capable of producing progesterone include reducing the activity of one or more enzymes that consume one or more progesterone pathway precursors (e.g., phytosterols). In certain embodiments, the one or more enzymes are involved in the phytosterol degradation pathway. In certain embodiments, the activity of one or more such enzymes is reduced by regulating the expression or activity of native enzymes. For example, the activity of such enzymes can be reduced by replacing the corresponding native promoter with a less active or inactive promoter or by deleting the corresponding gene.

[0120] In certain embodiments, the microbial cells of the transformation described herein contain the destruction (e.g., deletion) of one or more genes in Table 8 in the host microbial cell genome. In certain embodiments, destruction is achieved by transforming the corresponding integration plasmid into the host microbial cell (e.g., Mycobacterium neoaurum NRRL B-3805). In certain embodiments, the one or more genes that are destroyed include smo1, smo2, smo3, smo4, cyp142, kstD, chsH3, or any combination thereof. In certain embodiments, the one or more genes that are destroyed include cyp142, smo3, smo2, and smo1. In certain embodiments, the one or more genes that are destroyed include cyp142, smo3, smo2, smo1, and smo4.

[0121] In various embodiments, engineering a progesterone-producing microbial cell to reduce precursor consumption (e.g., phytosterol degradation) results in an increase in progesterone titer of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or at least 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, 50-fold, 51-fold, 52-fold, 53-fold, 54-fold, 55-fold, 56-fold, 57-fold, 58-fold, 59-fold, 60-fold, 61-fold, 62-fold, 63-fold, 64- times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, or 1000 times. In various embodiments, the progesterone titer is increased in the range of 10-fold to 1000-fold, 20-fold to 500-fold, 50-fold to 400-fold, 10-fold to 300-fold, or any range bounded by any of the foregoing values. These increases are relative to the progesterone titer observed in a progesterone-producing microbial cell that does not include a genetic alteration that reduces precursor consumption (e.g., phytosterol degradation) as discussed herein. The reference cell may (but does not necessarily) have other genetic alterations intended to increase progesterone production.

[0122] In various embodiments, engineering progesterone-producing microbial cells to reduce precursor consumption (e.g., phytosterol degradation) results in a reduction in the titer of an unwanted byproduct (e.g., androstenedione) to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001%. In various embodiments, the titer of the unwanted byproduct (e.g., androstenedione) is reduced in the range of 0.001%-90%, 0.1%-50%, 1%-20%, or in any range bounded by any of the foregoing values. These reductions are determined relative to the titers of unwanted byproducts observed in progesterone-producing microbial cells that do not include the genetic alterations discussed herein that reduce precursor consumption (e.g., phytosterol degradation). Such reference cells may (but need not) have other genetic alterations intended to increase progesterone production.

[0123] In various embodiments, the progesterone titer achieved by reducing precursor consumption (e.g., phytosterol degradation) is at least 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, or 900 mg / L, or at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 g / L. In various embodiments, the titer range is 10 mg / L to 200 g / L, 100 mg / L to 200 g / L, 1 g / L to 200 g / L, 1 g / L to 150 g / L, 1 g / L to 100 g / L, 1 g / L to 70 g / L, 1 g / L to 50 g / L, 5 g / L to 200 g / L, 5 g / L to 150 g / L, 5 g / L to 100 g / L, 5 g / L to 70 g / L, 5 g / L to 50 g / L g / L to 50 g / L, 10 g / L to 200 g / L, 10 g / L to 150 g / L, 10 g / L to 100 g / L, 10 g / L to 70 g / L, 10 g / L to 50 g / L, 20 g / L to 200 g / L, 20 g / L to 150 g / L, 20 g / L to 100 g / L, 20 g / L to 70 g / L, 20 g / L to 50 g / L, or any range defined by any of the above values.

[0124] Any of the above methods for increasing progesterone production can be combined to achieve higher progesterone production levels.

[0125] Ursodeoxycholic acid biosynthesis pathway

[0126] Ursodeoxycholic acid is a medication used to manage and treat cholestatic liver diseases. Most current production methods utilize chemical processes that require harsh chemicals and solvents. Furthermore, most current production methods rely on animal-derived bile acids. There are concerns that animal-derived bile acid products may contain animal pathogens such as prions and other harmful substances, including bacterial contaminants.

[0127] The present disclosure proposes a method for producing UDCA and other related compounds (e.g., 4-cholic acid-3-one, 3-keto-lithocholic acid and / or lithocholic acid) using plant sterols such as β-sitosterol as substrates. In addition, the method avoids any chemical steps. All steps can be completed by a single organism (e.g., Mycobacterium sp.) in a tank fermentation process. If one-step bioconversion proves difficult, there is also the opportunity to combine fermentation and biocatalysis to achieve the production of UDCA and other related compounds (e.g., 4-cholic acid-3-one, 3-keto-lithocholic acid and / or lithocholic acid).

[0128] Figure 8A A proposed biosynthetic pathway for converting plant sterols (e.g., β-sitosterol) to ursodeoxycholic acid is described. By disrupting the native bacterial sterol degradation pathway, plant sterols can be converted to 4-cholic acid-3-one (4CA3O) through partial degradation of the alkyl side chain. 4CA3O is then converted to 3-keto-lithocholic acid (3-keto-LCA) by 5β-steroid reductase (5βR) reducing the 4,5-double bond to form a 5β-reduced steroid. 3-keto-LCA is then converted to lithocholic acid (LCA) by 3α-hydroxysteroid dehydrogenase (3α-HSD) reducing the 3-position ketone to form the 3α-hydroxyl group. In the final step, LCA is converted to ursodeoxycholic acid (UDCA) by 7β-hydroxylase, which requires a redox chaperone protein (RPP) for activity.

[0129] Figure 8BA pathway for the degradation of phytosterols to androstenedione (AD) and androstenedione (ADD) has been described. This pathway begins by degradation of the side chains through a process similar to β-oxidation of fatty acids. Degradation is initiated by cytochrome P450 enzymes (cyp125, cyp142), which activate the side chains by oxidizing the terminal methyl group to a carboxylic acid. Subsequently, multiple enzymes act to convert this carboxylic acid intermediate into 3-oxo-4-ene-24-acyl-CoA (3-OCO-CoA). The side chain of 3-OCO-CoA is further degraded through two β-oxidation cycles to produce AD. The first step involves double bond formation by acyl-CoA dehydrogenase (chsE3). In the second step, the double bond is hydrated by hydratase (chsH3). Next, the resulting hydroxyl group is oxidized to a keto group by hydroxy-acyl-CoA dehydrogenase (hsd4A). The final β-oxidation step involves cleavage of the thiol group of coenzyme A from β-ketoacyl-CoA to form acetyl-CoA. This step is catalyzed by a thiolase (fadA5). The final round of β-oxidation involves another acyl-CoA dehydrogenase (chsE1 / chsE2), a hydrogenase (chsH1 / chsH2), and a thiolase (ltp2), leading to the formation of AD. 3-OCO-CoA 1-dehydrogenase (kstD) catalyzes the conversion of AD to ADD, forming a 1,2-double bond. Disruption of genes downstream of 3-OCO-CoA, particularly acyl-CoA dehydrogenases, chsE3, chsE1, and chsE, leads to the accumulation of 3-OCO-CoA. Hydrolysis of the coenzyme A group of 3-OCO-CoA by water or thioesterases results in the accumulation of 4CA3O. Disruption of other redundant or promiscuous acyl-CoA dehydrogenases may further increase 4CA3O levels.

[0130] Modification of microorganisms for the production of 4CA3O, 3-keto-LCA, LCA and / or UDCA

[0131] Methods for increasing UDCA production in microbial cells capable of producing UDCA include increasing the expression level or activity of one or more upstream enzymes that produce a precursor (e.g., 4CA3O); increasing the expression level or activity of one or more enzymes involved in converting 4CA3O to 3-keto-LCA; increasing the expression level or activity of one or more enzymes involved in converting 3-keto-LCA to LCA; and / or increasing the expression level or activity of one or more enzymes involved in converting LCA to UDCA. Figure 8A A schematic diagram of the metabolic pathway for converting phytosterols (e.g., β-sitosterol) to 4CA3O, 3-keto-LCA, LCA, and then to UDCA is shown. Suitable upstream pathway genes encoding these enzymes can be from any available source, including, for example, those disclosed herein. In certain embodiments, the one or more upstream enzymes include a thioesterase.

[0132] In certain embodiments, the activity of one or more upstream pathway enzymes is increased by regulating the expression or activity of native enzymes. For example, natural regulators of the expression or activity of such enzymes can be utilized to increase the activity of the appropriate enzymes. Additionally, one or more promoters can be substituted for the native promoter. In certain embodiments, the substituted promoter is more potent than the native promoter and / or is a constitutive promoter. In certain embodiments, one or more upstream pathway enzymes are expressed from an inducible promoter.

[0133] In certain embodiments, the activity of one or more upstream pathway enzymes is supplemented by introducing one or more corresponding genes into the modified microbial host cell. The upstream pathway genes introduced can be from an organism other than the host cell, or can simply be additional copies of the host's native genes. In certain embodiments, one or more such genes are introduced into a microbial host cell capable of producing 4CA3O, 3-keto-LCA, LCA and / or UDCA and expressed from a strong constitutive promoter (e.g., Pgl3) or an inducible promoter (Psmo3 or PkshA), and / or can preferably be codon-optimized to enhance expression in the selected microbial host cell.

[0134] Generally, genes encoding enzymes can be introduced and expressed into microbial cells using conventional genetic modification techniques, thereby allowing the modified microbial cells to have any active enzyme involved in the conversion of 4CA3O to 3-keto-LCA (e.g., 5β-steroid reductase (5βR)), enzymes involved in the conversion of 3-keto-LCA to LCA (e.g., 3α-hydroxysteroid dehydrogenase (3α-HSD)), and / or enzymes involved in the conversion of LCA to UDCA (e.g., 7β-hydroxylase and / or one or more redox chaperone proteins (e.g., ferredoxin and ferredoxin reductase, or cytochrome P450 reductase)). In certain embodiments, genetic alteration is achieved by transforming the corresponding expression vector into a host microbial cell (e.g., Mycobacterium neoaurum NRRL B-3805). In certain embodiments, each heterologous gene is located downstream of a ribosome binding site. In certain embodiments, expression of one or more heterologous genes is controlled by a strong constitutive promoter (e.g., Pgl3). In certain embodiments, the heterologous gene is expressed under the control of an inducible promoter (eg, Psmo3 or PkshA). The heterologous gene may preferably be codon-optimized to enhance expression in the selected microbial host cell.

[0135] Generally, any active 5β-steroid reductase (5βR), 3α-hydroxysteroid dehydrogenase (3α-HSD), 7β-hydroxylase and / or one or more redox chaperones (e.g., ferredoxin and ferredoxin reductase, or cytochrome P450 reductase) can be introduced and expressed in the microbial cell by conventional genetic engineering techniques. Suitable 5β-steroid reductase (5βR), 3α-hydroxysteroid dehydrogenase (3α-HSD), 7β-hydroxylase and / or one or more redox chaperones can be derived from any source, including plants, archaea, fungi, gram-positive bacteria and gram-negative bacteria.

[0136] One or more copies of any of these genes can be introduced into the selected microbial host cell. If more than one gene copy is introduced, these copies can have the same or different nucleotide sequences. In certain embodiments, one or two (or all) heterologous genes are expressed by a strong constitutive promoter (e.g., Pgl3). In certain embodiments, the heterologous genes are expressed by an inducible promoter (e.g., Psmo3 or PkshA). The heterologous genes can preferably be codon-optimized to enhance expression in the selected microbial host cell.

[0137] In certain embodiments, the gene ID, source, and amino acid sequence of the 5β-steroid reductase (5βR) described herein are listed in the table below.

[0138] Table 4 5βR used in this disclosure

[0139]

[0140]

[0141] In certain embodiments, the gene ID, source, and amino acid sequence of the 3α-hydroxysteroid dehydrogenase (3α-HSD) described herein are listed in the table below.

[0142] Table 5 3α-HSD used in this disclosure

[0143]

[0144]

[0145] In certain embodiments, the gene ID, source, and amino acid sequence of the 7β-hydroxylases described herein are listed in the table below.

[0146] Table 6 7β-hydroxylases used in the present disclosure

[0147]

[0148]

[0149]

[0150] In certain embodiments, the gene ID, source, and amino acid sequence of the ferredoxin proteins described herein are listed in Table 2. In certain embodiments, the gene ID, source, and amino acid sequence of the ferredoxin reductases described herein are listed in Table 3.

[0151] In certain embodiments, the non-native 5βR has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: 73-97. In certain embodiments, the non-native 3α-HSD has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: 98-108. In certain embodiments, the non-native 7β-hydroxylase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: 109-135. In certain embodiments, the non-native ferredoxin protein has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to any one of SEQ ID NOs: 5, 25-37, and 137. In certain embodiments, the non-native ferredoxin reductase has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to any one of SEQ ID NOs: 6, 38-46, and 136.

[0152] In certain embodiments, the modified microbial cells described herein express non-native 5βR (e.g., any 5βR enzyme in Table 4). In certain embodiments, the modified microbial cells described herein express non-native 3α-HSD (e.g., any 3α-HSD enzyme in Table 5). In certain embodiments, the modified microbial cells described herein express non-native 7β-hydroxylase (e.g., any 7β-hydroxylase in Table 6). In certain embodiments, the modified microbial cells described herein express one or more non-native redox chaperones, such as non-native ferredoxin, non-native ferredoxin reductase, or both. In certain embodiments, the non-native ferredoxin is any one of the ferredoxins in Table 2. In certain embodiments, the non-native ferredoxin is a Synechocystis sp. ferredoxin (e.g., FDX_SYNY4_2). In certain embodiments, the non-native ferredoxin reductase is any one of the ferredoxin reductases in Table 3. In certain embodiments, the non-native ferredoxin reductase is a Synechocystis sp. ferredoxin or a derivative thereof (eg, FDXR_SYNY4_2).

[0153] In certain embodiments, the non-native state 5βR can increase the production of 3-keto-LCA, LCA and / or UDCA by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, or at least 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, 50-fold, 51-fold, 52-fold, 53-fold, 54-fold, 55-fold, 56-fold, 57-fold, 58-fold, 59-fold, 60-fold, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times or 1000 times.

[0154] In certain embodiments, the non-native 3α-HSD can increase the production of LCA and / or UDCA by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, or by at least 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, 50-fold, 51-fold, 52-fold, 53-fold, 54-fold, 55-fold, 56-fold, 57-fold, 58-fold, 59-fold, 60-fold, 61-fold, 62-fold, 63-fold, 64-fold, 65-fold times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, or 1000 times.

[0155] In certain embodiments, the non-native 7β-hydroxylase can increase the production of UDCA by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, or at least 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, 50-fold, 51-fold, 52-fold, 53-fold, 54-fold, 55-fold, 56-fold, 57-fold, 58-fold, 59-fold, 60-fold, 8 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, or 1000 times.

[0156] In certain embodiments, the non-native ferredoxin and / or non-native ferredoxin reductase can increase the production of UDCA by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or at least 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, 50-fold, 51-fold, 52-fold, 53-fold, 54-fold, 55-fold, 56-fold, 57-fold, 58-fold, 59-fold, 60- 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times or 1000 times.

[0157] In one aspect, the modified microbial cells described herein contain one or more vectors suitable for expressing proteins (e.g., any combination of 5βR, 3α-HSD, 7β-hydroxylase, and redox chaperone proteins) in host microbial cells. In certain embodiments, the vectors include one or more gene sequences encoding the proteins described herein. The gene sequences can be derived from any of the source genes or source organisms listed in Tables 2-6.

[0158] In certain embodiments, the modified microbial cells described herein contain a first vector and a second vector. In certain embodiments, the first vector described herein comprises, from the 5' end to the 3' end, or in any order: an inducible promoter (e.g., Psmo3), a first gene sequence encoding any 7β-hydroxylase listed in Table 6, a second gene sequence encoding any ferredoxin listed in Table 2, and a third gene sequence encoding any ferredoxin reductase listed in Table 3. In certain embodiments, each of the first gene sequence, the second gene sequence, and the third gene sequence has a ribosome binding site upstream. In certain embodiments, the second vector described herein comprises, from the 5' end to the 3' end, or in any order: a constitutive promoter (e.g., Pgl3) or an inducible promoter (e.g., PkshA), a first gene sequence encoding any 5βR listed in Table 4, and a second gene sequence encoding any 3α-HSD listed in Table 5. In certain embodiments, each of the first gene sequence and the second gene sequence has a ribosome binding site upstream.

[0159] In certain embodiments, the first vector comprises a Psmo3 promoter, a first gene sequence encoding Allokutzneria albata 7β-hydroxylase (e.g., SEQ ID NO: 109), a second gene sequence encoding a Synechocystis sp. ferredoxin (SEQ ID NO: 137), and a third gene sequence encoding a ferredoxin reductase from Synechocystis sp. (e.g., SEQ ID NO: 136); and the second vector comprises a PkshA promoter, a first gene sequence encoding Tarenaya hassleriana 5βR (e.g., SEQ ID NO: 77), and a second gene sequence encoding Eggerthella sp. 3α-HSD (SEQ ID NO: 99).

[0160] In certain embodiments, the first vector comprises a Psmo3 promoter, a first gene sequence encoding Allokutzneria albata 7β-hydroxylase (e.g., SEQ ID NO: 109), a second gene sequence encoding a Synechocystis sp. ferredoxin (SEQ ID NO: 137), and a third gene sequence encoding a ferredoxin reductase from Synechocystis sp. (e.g., SEQ ID NO: 136); and the second vector comprises a PkshA promoter, a first gene sequence encoding Tarenaya hassleriana 5βR (e.g., SEQ ID NO: 77), and a second gene sequence encoding Ruminococcus gnavus 3α-HSD (SEQ ID NO: 104).

[0161] In certain embodiments, the first vector comprises a Psmo3 promoter, a first gene sequence encoding Allokutzneria albata 7β-hydroxylase (e.g., SEQ ID NO: 109), a second gene sequence encoding Synechocystis sp. ferredoxin (SEQ ID NO: 137), and a third gene sequence encoding a ferredoxin reductase from Synechocystis sp. (e.g., SEQ ID NO: 136); and the second vector comprises a Pg13 promoter, a first gene sequence encoding Tarenaya hassleriana 5βR (e.g., SEQ ID NO: 77), and a second gene sequence encoding Eggerthella sp. 3α-HSD (SEQ ID NO: 99).

[0162] In certain embodiments, the first vector comprises a Psmo3 promoter, a first gene sequence encoding Allokutzneria albata 7β-hydroxylase (e.g., SEQ ID NO: 109), a second gene sequence encoding Synechocystis sp. ferredoxin (SEQ ID NO: 137), and a third gene sequence encoding a ferredoxin reductase from Synechocystis sp. (e.g., SEQ ID NO: 136); and the second vector comprises a PkshA promoter, a first gene sequence encoding Raphanus sativus 5βR (e.g., SEQ ID NO: 95), and a second gene sequence encoding Eggerthella sp. 3α-HSD (SEQ ID NO: 99).

[0163] In certain embodiments, the first vector comprises a Psmo3 promoter, a first gene sequence encoding Allokutzneria albata 7β-hydroxylase (e.g., SEQ ID NO: 109), a second gene sequence encoding Synechocystis sp. ferredoxin (SEQ ID NO: 137), and a third gene sequence encoding a ferredoxin reductase from Synechocystis sp. (e.g., SEQ ID NO: 136); and the second vector comprises a Pg13 promoter, a first gene sequence encoding Tarenaya hassleriana 5βR (e.g., SEQ ID NO: 77), and a second gene sequence encoding Ruminococcus gnavus 3α-HSD (SEQ ID NO: 104).

[0164] In various embodiments, the 4CA3O, 3-keto-LCA, LCA and / or UDCA titer is increased in the range of 10-fold to 1000-fold, 20-fold to 500-fold, 50-fold to 400-fold, 10-fold to 300-fold, or any range defined by any of the foregoing values. These increases are relative to the 4CA3O, 3-keto-LCA, LCA and / or UDCA titer observed in a microbial cell that produces 4CA3O, 3-keto-LCA, LCA and / or UDCA but does not include the genetic alterations discussed herein. The reference cell may (but does not necessarily) have other genetic alterations intended to increase 4CA3O, 3-keto-LCA, LCA and / or UDCA production.

[0165] In various embodiments, a titer of at least 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, or 900 mg / L, or at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 g / L of 4CA3O, 3-keto-LCA, LCA, and / or UDCA is achieved by expressing a non-native 5βR, a non-native 3α-HSD, a non-native 7β-hydroxylase, and one or more non-native redox chaperone proteins. In various embodiments, the titer range is 10 mg / L to 200 g / L, 100 mg / L to 200 g / L, 1 g / L to 200 g / L, 1 g / L to 150 g / L, 1 g / L to 100 g / L, 1 g / L to 70 g / L, 1 g / L to 50 g / L, 5 g / L to 200 g / L, 5 g / L to 150 g / L, 5 g / L to 100 g / L, 5 g / L to 70 g / L, 5 g / L to 50 g / L, g / L to 50 g / L, 10 g / L to 200 g / L, 10 g / L to 150 g / L, 10 g / L to 100 g / L, 10 g / L to 70 g / L, 10 g / L to 50 g / L, 20 g / L to 200 g / L, 20 g / L to 150 g / L, 20 g / L to 100 g / L, 20 g / L to 70 g / L, 20 g / L to 50 g / L, or any range defined by any of the above values.

[0166] In certain embodiments, microbial cells producing 4CA3O, 3-keto-LCA, LCA and / or UDCA are engineered to overexpress a thioesterase (either native or non-native) to convert 4CA3O to 3-keto-LCA, convert 3-keto-LCA to LCA, and / or convert LCA to UDCA, which can increase the titer of 4CA3O, 3-keto-LCA, LCA and / or UDCA by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, or at least 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, 50-fold, 51-fold, 52-fold, 53 8 times, 8.5 times, 9 times, 9.5 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times or 1000 times. In various embodiments, the titer of 4CA3O, 3-keto-LCA, LCA and / or UDCA is increased in the range of 10-fold to 1,000-fold, 20-fold to 500-fold, 50-fold to 400-fold, 10-fold to 300-fold, or any range defined by any of the foregoing values. These increases are relative to the titer of 4CA3O, 3-keto-LCA, LCA and / or UDCA observed in a microbial cell that produces 4CA3O, 3-keto-LCA, LCA and / or UDCA but does not include the genetic alterations discussed herein. The reference cell may (but does not necessarily) have other genetic alterations intended to increase 4CA3O, 3-keto-LCA, LCA and / or UDCA production.

[0167] In various embodiments, the titer of UDCA or its precursor (e.g., 4CA3O, 3-keto-LCA and / or LCA) is at least 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 600, 750, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7500, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 75000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 750000, 800000, 90000, 1000000, 2000000, 3000000, 400000, 500000, 600000, 750000, 800000 140, 150, 160, 170, 180, 190, 200 or 300 g / L, or at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or 300 g / L. In various embodiments, the titer ranges from 10 mg / L to 300 g / L, 100 mg / L to 300 g / L, 1 g / L to 300 g / L, 1 g / L to 200 g / L, 1 g / L to 150 g / L, 1 g / L to 120 g / L, 1 g / L to 90 g / L, 1 g / L to 70 g / L, 5 g / L to 300 g / L, 5 g / L to 200 g / L, 5 g / L to 150 g / L, 5 g / L to 120 g / L, 5 g / L to 90 g / L, 5 g / L to 90 g / L, g / L, 10 g / L to 70 g / L, 10 g / L to 300 g / L, 10 g / L to 200 g / L, 10 g / L to 150 g / L, 10 g / L to 120 g / L, 10 g / L to 90 g / L, 10 g / L to 70 g / L, 20 g / L to 300 g / L, 20 g / L to 200 g / L, 20 g / L to 150 g / L, 20 g / L to 120 g / L, 20 g / L to 90 g / L, 20 g / L to 70 g / L, or any range defined by any of the above values.

[0168] Any of the above methods for increasing the production of 4CA3O, 3-keto-LCA, LCA and / or UDCA can be combined in any manner to achieve higher production levels of 4CA3O, 3-keto-LCA, LCA and / or UDCA.

[0169] Reduce consumption of UDCA and / or its precursors

[0170] Methods for increasing UDCA production and reducing unwanted byproducts in microbial cells capable of producing UDCA include reducing the activity of one or more enzymes that consume one or more UDCA pathway precursors (e.g., 4-cholic acid-3-one). In certain embodiments, the one or more enzymes are involved in the phytosterol degradation pathway. In certain embodiments, the activity of one or more such enzymes is reduced by regulating the expression or activity of native enzymes. For example, the activity of such enzymes can be reduced by replacing the native promoter of the corresponding gene with a less active or inactive promoter or by deleting the corresponding gene.

[0171] In certain embodiments, the microbial cell of transformation as herein described contains the destruction (for example, deletion) of one or more genes in Table 11 and Table 13 in the host microbial cell genome.In certain embodiments, by corresponding integration plasmid transformation into host microbial cell (for example, new gold mycobacterium (Mycobacterium neoaurum) NRRL B-3805) realize destruction.In certain embodiments, described one or more genes that are destroyed comprise chsE3, chsE1, chsE2, casC, chsH3, hsd4A, casC3, kstD, chsE5, chsE4, fadD17 or its any combination.In certain embodiments, described one or more genes that are destroyed comprise chsE3, chsE1, chsE2 and casC3.In certain embodiments, described one or more genes that are destroyed comprise chsE3, chsE1, chsE2, casC3, kstD and chsH3. In certain embodiments, the one or more genes disrupted include chsE3, chsE1, chsE2, casC3, kstD, chsH3, and chsE5. In certain embodiments, the one or more genes disrupted include chsE3, chsE1, chsE2, casC3, kstD, chsH3, chsE4, and fadD17. In certain embodiments, the one or more genes disrupted include chsE3, chsE1, chsE2, casC3, kstD, chsH3, chsE4, chsE5, and fadD17.

[0172] In certain embodiments, the microbial cells of the transformation described herein contain one or more genes encoding the native enzymes that reduce 4CA3O accumulation (e.g., deletion). In certain embodiments, the destruction is achieved by transforming the corresponding integration plasmid into the host microbial cell (e.g., Mycobacterium neoaurum NRRL B-3805). In certain embodiments, the native enzymes include acyl-CoA synthetase, acyl-CoA ligase, acyl-Co A reductase, aldehyde reductase, or any combination thereof. In certain embodiments, the one or more genes destroyed as described herein include fadD17.

[0173] In certain embodiments, the microbial cells of the transformation described herein contain one or more genes encoding enzymes that cause unwanted by-products (e.g., androstenedione (AD), androstenedione (ADD) and / or biredo). In certain embodiments, the destruction is achieved by transforming the corresponding integration plasmid into the host microbial cell (e.g., Mycobacterium neoaurum NRRL B-3805). In certain embodiments, the one or more genes that are destroyed include kstD, chsH3, hsd4A, ltp2, or any combination thereof. In certain embodiments, the native enzyme destroyed as described herein includes 3-ketosteroid-δ-1-dehydrogenase.

[0174] In certain embodiments, the modified microbial cells described herein contain one or more genes encoding the native enzymes that reduce the accumulation of 3-keto-LCA, LCA and / or UDCA (e.g., deletion). In certain embodiments, the destruction is achieved by transforming the corresponding integration plasmid into a host microbial cell (e.g., Mycobacterium neoaurum NRRL B-3805). In certain embodiments, the native enzyme participates in the oxidation of the 3α-hydroxyl group and the oxidation of the 5-carbon position to form a double bond between the 4 and 5 positions of LCA and / or UDCA, or participates in the oxidation, epimerization, or dehydroxylation of the 7β-hydroxyl group of UDCA.

[0175] In various embodiments, engineering microbial cells producing 4CA3O, 3-keto-LCA, LCA and / or UDCA to reduce precursor consumption (e.g., phytosterol degradation), increase 4CA3O, 3-keto-LCA, LCA and / or UDCA accumulation, and / or reduce accumulation of undesirable byproducts can increase 4CA3O, 3-keto-LCA, LCA and / or UDCA titer by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, or at least 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, 50-fold, 51-fold, 52-fold, 53-fold, 54-fold, 55-fold, times, 8.5 times, 9 times, 9.5 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, or 1000 times. In various embodiments, the titer of 4CA3O, 3-keto-LCA, LCA and / or UDCA is increased in a range of 10-fold to 1000-fold, 20-fold to 500-fold, 50-fold to 400-fold, 10-fold to 300-fold, or in any range defined by any of the foregoing values. These increases are relative to the titer of 4CA3O, 3-keto-LCA, LCA and / or UDCA observed in a microbial cell that produces 4CA3O, 3-keto-LCA, LCA and / or UDCA but does not include the genetic alterations discussed herein. The reference cell may (but does not necessarily) have other genetic alterations intended to increase 4CA3O, 3-keto-LCA, LCA and / or UDCA production.

[0176] In various embodiments, microbial cells producing 4CA3O, 3-keto-LCA, LCA and / or UDCA are engineered to reduce precursor consumption (e.g., phytosterol degradation), increase 4CA3O, 3-keto-LCA, LCA and / or UDCA accumulation, and / or reduce accumulation of undesirable byproducts, such that the titer of the undesirable byproduct (e.g., AD, ADD and / or biredo) is reduced to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.3%, 0.2%, 0.1%, 0.05%, 0.01%, 0.005% or 0.001%. In various embodiments, the titer of an unwanted byproduct (e.g., AD, ADD, and / or biredo) is reduced in the range of 0.001%-90%, 0.1%-50%, 1%-20%, or any range bounded by any of the above values. These reductions are relative to the titer of the unwanted byproduct observed in a microbial cell producing 4CA3O, 3-keto-LCA, LCA, and / or UDCA that does not include the genetic alterations discussed herein. The reference cell may (but does not necessarily) have other genetic alterations intended to increase UDCA production.

[0177] In various embodiments, the titer of 4CA3O, 3-keto-LCA, LCA and / or UDCA or its precursors (e.g., 4CA3O, 3-keto-LCA and / or LCA) is at least 10, 20, 30, 40, 50, 75, ... 140, 150, 160, 170, 180, 190, 200, or 300 g / L. In various embodiments, the titer range is 10 mg / L to 300 g / L, 100 mg / L to 300 g / L, 1 g / L to 300 g / L, 1 g / L to 200 g, 1 g / L to 150 g / L, 1 g / L to 120 g / L, 1 g / L to 90 g / L, 1 g / L to 70 g / L, 5 g / L to 300 g / L, 5 g / L to 200 g / L, 5 g / L to 150 g / L, 5 g / L to 120 g / L, 5 g / L to 90 g / L, 5 g / L to 70 g / L. 0 g / L, 10 g / L to 300 g / L, 10 g / L to 200 g / L, 10 g / L to 150 g / L, 10 g / L to 120 g / L, 10 g / L to 90 g / L, 10 g / L to 70 g / L, 20 g / L to 300 g / L, 20 g / L to 200 g / L, 20 g / L to 150 g / L, 20 g / L to 120 g / L, 20 g / L to 90 g / L, 20 g / L to 70 g / L, or any range bounded by any of the foregoing values ​​for microbial host cells

[0178] Any microorganism that can be used to express the introduced gene can be modified for the fermentative production of progesterone, 4CA3O, 3-keto-LCA, LCA and / or UDCA as described above. In certain embodiments, the microorganism is a microorganism that is naturally unable to fermentatively produce progesterone, 4CA3O, 3-keto-LCA, LCA and / or and / or UDCA. In certain embodiments, the microorganism is an easily cultured microorganism, for example, a microorganism known to be used as a host cell in the fermentative production of the target compound. Bacterial cells, including Gram-positive or Gram-negative bacteria, can be modified as described above. Examples include Corynebacterium glutamicum, Bacillus subtilis, and Bacillus cereus. subtilus), B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, B. thuringiensis, S. albus, S. lividans, S. coelicolor, S. griseus, Pseudomonas sp. (e.g., Pseudomonas putida), putida), Pseudomonas alcaligenes, P. citrea, Lactobacillus spp. (e.g., Lactococcus lactis, L. plantarum), Listeria grayi, E. coli, E. faecium, E. gallinarum, E. casseliflavus, and / or E. faecalis cells.

[0179] In certain embodiments, microorganism as herein described or microbial cell are mycobacteria (Mycobacterium) strains.In certain embodiments, microorganism as herein described or microbial cell are new gold mycobacteria (Mycobacterium neoaurum) (for example, new gold mycobacteria NRRL B-3805).In certain embodiments, microorganism as herein described or microbial cell are mycobacteria (Mycobacterium spp.) strains (for example, mycobacterium smegmatis (Mycolicibacter smegmatis), new gold mycobacteria (Mycobacterium neoaurum) (new gold mycobacteria (Mycolicibacterium neoaurum)) or fortuitum mycobacterium (Mycolicibacterium fortuitum)). In certain embodiments, the microorganisms or microbial cells described herein are Rhodococcus spp. strains (e.g., Rhodococcus jostii, Rhodococcus rhodochrous, Rhodococcus erythropolis, Rhodococcus jostii, or Rhodococcus ruber). In certain embodiments, the microorganisms or microbial cells described herein are Nocardia spp. strains (e.g., Nocardia corallina, Nocardia corallina, or Nocardia opaca).

[0180] There are many types of anaerobic cells that can be used as the microbial host cells in the methods described herein. In certain embodiments, the microbial cells are obligate anaerobic cells. Under conditions in which oxygen is present (if any), obligate anaerobes cannot usually grow well. Of course, a small amount of oxygen can be present, that is, obligate anaerobes have a certain degree of tolerance to low levels of oxygen. The obligate anaerobes transformed as described above can be grown under essentially anaerobic conditions, wherein the amount of oxygen present is harmless for the growth, maintenance and / or fermentation of the anaerobic bacteria.

[0181] Alternatively, the microbial host cells used in the methods described herein can be facultative anaerobic cells. If there is oxygen, facultative anaerobic bacteria can produce cellular ATP by aerobic respiration (e.g., utilizing the TCA cycle). However, facultative anaerobic bacteria can also grow in the absence of oxygen. Facultative anaerobic bacteria modified as described above can be grown under essentially anaerobic conditions, wherein the amount of oxygen present is harmless to the growth, maintenance, and / or fermentation of the anaerobic bacteria, or can be grown in the presence of a greater amount of oxygen.

[0182] In certain embodiments, the microbial host cells used in the methods described herein are filamentous fungal cells (see, e.g., Berka & Barnett, Biotechnology Advances, (1989), 7(2): 127-154). Examples include Trichoderma longibrachiatum, T. viride, T. koningii, T. harzianum, Penicillium sp., Humicola insolens, H. lanuginose, H. grisea, Chrysosporium sp., C. lucknowense, Gliocladium sp., Aspergillus sp. (e.g., A. oryzae, A. niger, A. sojae, A. japonicus, A. nidulans, or A. awamori), Fusarium sp. sp.) (e.g., F. roseum, F. graminum, F. grainis, F. oxysporuim, or F. venenatum)), Neurospora sp. (e.g., N. crassa or Hypocrea sp.), Mucor sp. (e.g., M. miehei), Rhizopus sp., and Emericella sp. cells. In a specific embodiment, the fungal cell of the above transformation is Aspergillus nidulans (A. nidulans), Aspergillus awamori (A. awamori), Aspergillus oryzae (A. oryzae), Aspergillus aculeatus (A. aculeatus), Aspergillus niger (A. niger), Aspergillus japonicus (A. japonicus), Trichoderma reesei (T. reesei), Trichoderma viride (T. viride), Fusarium oxysporum (F. oxysporuim) or Fusarium solani (F. solani). Exemplary plasmids or plasmid components used with such hosts include those described in U.S. Patent Publication No. 2007 / 012013.

[0183] Yeast can also be used as a microbial host cell in the methods described herein. Examples include: Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., Hansenula polymorpha, Pichia stipites, Kluyveromyces marxianus, Kluyveromyces spp., Yarrowia lipolytica, and Candida sp. In certain embodiments, the Saccharomyces sp. is Saccharomyces cerevisiae (see, e.g., Romanos et al., Yeast, (1992), 8(6): 423-488). Exemplary plasmids or plasmid components for use with such hosts include those described in US Patent No. 7,659,097 and US Patent Publication No. 2011 / 0045563.

[0184] In certain embodiments, the host cell can be an algal cell derived from, for example, green algae, red algae, glaucophyte, divinophyte, euglenoid, chromophore, or dinoflagellate (see, e.g., Saunders & Warmbrodt, "Gene Expression in Algae and Fungi, Including Yeast," (1993), National Agricultural Library, Beltsville, Md.). Exemplary plasmids or plasmid components for algal cells include those described in U.S. Patent Publication No. 2011 / 0045563.

[0185] In other embodiments, the host cell is a cyanobacterial, for example, any of the cyanobacteria classified based on morphology into the following groups: Chlorococcales, Pleurocapsales, Oscillatoriales, Nostocales, Synechosystic, or Stigonematales (see, e.g., Lindberg et al., Metab. Eng. (2010) 12(1): 70-79). Exemplary plasmids or plasmid components for cyanobacterial cells include those described in U.S. Patent Publications 2010 / 0297749 and 2009 / 0282545 and International Patent Publication WO2011 / 034863.

[0186] In other embodiments, host cells can include other prokaryotes and eukaryotes, but are not limited to bacteria or yeast. Exemplary species include Escherichia coli., Saccharomyces cerevisiae, Saccharomyces kluyveri, Candida boidinii, Clostridium kluyveri, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum, Clostridium perfringens, Clostridium difficile, Clostridium botulinum, Clostridium tyrobutyricum, Clostridium tetanomorphum, Clostridium tetani, Clostridium propionate, Clostridium tetanomorphum, Clostridium tetani ... propionicum), Clostridium aminobutyricum, Clostridium subterminale, Clostridium sticklandii, Ralstonia eutropha, Mycobacterium bovis, Mycobacterium tuberculosis, Porphyromonas gingivalis, Arabidopsis thaliana, Thermus thermophilus, Pseudomonas species, including Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas astutzeri, Pseudomonas fluorescens, Homo sapiens sapiens), Oryctolagus cuniculus, Rhodobacterspaeroides), Thermoanaerobacter brockii, Metallosphaera sedula, Leuconostoc mesenteroides, Chloroflexus aurantiacus, Roseiflexus castenholzii, Erythrobacter, Simmondsia chinensis, Acinetobacter species, including Acinetobacter calcoaceticus and Acinetobacter baylyi, Porphyromonas gingivalis, Sulfolobus tokodaii, Sulfolobus solfataricus, Sulfolobus acidocaldarius, Bacillus subtilis subtilis), Bacillus cereu, Bacillus megaterium, Bacillus brevis, Bacillus pumilus, Rattus norvegicus, Klebsiella pneumonia, Klebsiella oxytoca, Euglena gracilis, Treponema denticola, Moorella thermoacetica, Thermotogamaritima, Halobacterium salinarum, Geobacillus stearothermophilus, Aeropyrum pernix, Sus scrofa, Caenorhabditis elegans, Corynebacterium glutamicum glutamicum), Acidaminococcus fermentans, Lactococcus lactislactis, Lactobacillus plantarum, Streptococcus thermophilus, Enterobacter aerogenes, Candida, Aspergillus terreus, Pedicoccus pentosaceus, Zymomonas mobilus, Acetobacter pasteurians, Kluyveromyces lactis, Eubacterium barkeri, Bacteroides capillosus, Anaerotruncus colihominis, Natranaerobius thermophilus, Campylobacter jejuni, Haemophilus influenzae, Serratia marcescens, Citrobacter amalonicid amalonaticus), Myxococcus xanthus, Fusobacterium nuleatum, Penicillium chrysogenum, Nocardia iowensis, Nocardia farcinica, Streptomyces griseus, Schizosaccharomyces pombe, Geobacillus thermoglucosidase, Salmonella typhimurium, Vibrio cholerae, Heliobacter pylori, Nicotiana tabacum, Oryza sativa, Haloferax mediterranei, Agrobacterium tumefaciens, Achromobacter denitrificans denitrificans), Fusobacterium nucleatumnucleatum), Streptomyces clavuligerus, Acinetobacter baumanii, Mus musculus, Lachance akluyveri, Trichomonas vaginalis, Trypanosoma brucei, Pseudomonas stutzeri, Bradyrhizobium japonicum, Mesorhizobium loti, Bos taurus, Nicotiana glutinosa, Vibrio vulnificus, Selenomonas ruminantium, Vibrio parahaemolyticus, Archaeoglobus fulgidus, Haloarcula marismortui), Pyrobacula erophilum, Mycobacterium smegmatis MC2155, Mycobacterium avium subsp. Paratuberculosis K-10, Mycobacterium marinum M, Tsukamurella paurometabola DSM20162, Cyanobium PCC7001, and Dictyostelium discoideum AX4.

[0187] In certain embodiments, suitable organisms include Acinetobacter baumannii Naval-82, Acinetobacter sp. ADPl, Acinetobacter sp. strain M-1, Actinobacillus succinogenes 130Z, Allochromatium vinosum DSM180, Amycolatopsis methanolica, Arabidopsis thaliana, Atopobium parvulum DSM20469, Azotobacter vinelandii DJ, Bacillus alcalophilus ATCC27647, Bacillus azotoformans LMG9581, Bacillus coagulans, and Bacillus thaliana. coagulans 36D1, Bacillus megaterium, Bacillus methanolicus MGA3, Bacillus methanolicus PB1, Bacillus methanolicus PB-1, Bacillus sselenitireducens MLS10, Bacillus smithii, Bacillus subtilis, Burkholderia cenocepacia, Burkholderia cepacia, Burkholderia multivorans, Burkholderia pyrrocinia, Burkholderia stabilis, Burkholderia thailandensis E264, Burkholderia bacterium)Joshi_001, Campylobacter jejuni, Candida albicans, Candida boidiniiboidinii), Candida methylica, Carboxydothermus hydrogenoformans, Carboxydothermus hydrogenoformans Z-2901, Caulobacter sp. AP07, Chloroflexus aggregans DSM 9485, Chloroflexus aurantiacus J-10-fl, Citrobacter freundii, Citrobacter koseri ATCC BAA-895, Citrobacter youngae, Clostridium, Clostridium acetobutylicum, Clostridium acetobutylicum ATCC 824, Clostridium aciduriae acidurici), Clostridium aminobutyricum, Clostridium asparagiforme DSM 15981, Clostridium beijerinckii, Clostridium beijerinckii NCIMB 8052, Clostridium bolteae ATCC BAA-613, Clostridium carboxidivorans P7, Clostridium cellulovorans 743B, Clostridium difficile, Clostridium hiranonis DSM 13275, Clostridium hylemonae DSM 15053, Clostridium kluyveri, Clostridium kluyveri DSM 555, Clostridium ljungdahli, Clostridium ljungdahli DSM 13528, Clostridium methylpentosaceaemethylpentosum DSM5476, Clostridium pasteurianum, Clostridium pasteurianum DSM525, Clostridium perfringens, Clostridium perfringens ATCC 13124, Clostridium perfringens str.13, Clostridium phytofermentans ISDg, Clostridium saccharobutylicum, Clostridium saccharoperbutylacetonicum, Clostridium saccharoperbutylacetonicum N1-4, Clostridium tetani, Corynebacterium glutamicum ATCC 14067, Corynebacterium glutamicum R, Corynebacterium sp.) U-96, Corynebacterium variabile, Cupriavidus necator N-1, Cyanobium PCC7001, Desulfatibacillum alkenivorans AK-01, Desulfitobacterium hafniense, Desulfitobacterium metallireducens DSM 15288, Desulfotomaculum reducens MI-1, Desulfovibrio africanus str. Walvis Bay, Desulfovibrio fructosovorans JJ, Desulfovibrio vulgaris str. Hildenborough, Desulfovibrio vulgaris str. 'Miyazaki F', Dictyostelium discoideum AX4, Escherichia coli, Escherichia coli)K-12, Escherichia colicoli K-12MG1655, Eubacterium hallii DSM 3353, Flavobacterium frigoris, Fusobacterium nucleatum subsp. Polymorphum ATCC 10953, Geobacillus sp. Y4.1MC1, Geobacillus thermodenitrificans NG80-2, Geobacter bemidjiensis Bem, Geobacter sulfurreducens, Geobacter sulfurreducens PCA, Geobacillus stearothermophilus DSM 2334, Haemophilus influenzae, Helicobacter pylori, Homo sapiens sapiens), Hydrogenobacter thermophilus, Hydrogenobacter thermophilus TK-6, Hyphomicrobium denitrificans ATCC 51888, Hyphomicrobium zavarzinii, Klebsiella pneumoniae, Klebsiella pneumoniae subsp. Pneumoniae MGH 78578, Lactobacillus brevis ATCC 367, Leuconostoc mesenteroides, Lysinibacillus fusiformis, Lysinibacillus sphaericus, Mesorhizobium nigrum loti)MAFF303099, Metallosphaerasedula, Methanosarcina acetivorans, Methanosarcina acetivorans C2A, Methanosarcinabarkeri), Methanosarcina mazei Tuc01, Methylobacter marinus, Methylobacterium extorquens, Methylobacterium extorquens AM1, Methylococcus capsulatas, Methylomonas aminofaciens, Moorellathernoacetica, Mycobacterium sp. strain JC1 DSM 3803, Mycobacterium avium subsp. Paratuberculosis K-10, Mycobacterium bovis BCG, Mycobacterium gastri, Mycobacterium marinum M, Mycobacterium smegmatis, Mycobacterium smegmatis smegmatis MC2 155, Mycobacterium tuberculosis, Nitrosopumilus solaria BD31, Nitrososphaera gargensis Ga9.2, Nocardia farcinica IFM 10152, Nocardia iowensis (sp. NRRL 5646), Nostoc sp. PCC 7120, Ogataea angusta, Ogataea parapolymorpha DL-1, Hansenula polymorpha DL-1, Paenibacillus peoriae KCTC 3763, Paracoccus denitrificans, Penicillium chrysogenum), Photobacterium profundum 3TCK, Phytofermentans ISDg, Pichia pastoris, Picrophilus torridus DSM9790, Porphyromonas gingivalisgingivalis), Porphyromonas gingivalis W83, Pseudomonas aeruginosa PA01, Pseudomonas denitrificans, Pseudomonas knackmussii, Pseudomonas putida, Pseudomonas sp., Pseudomonas syringaepv.Syringae B728a, Pyrobaculum islandicum DSM 4184, Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii OT3, Ralstonia eutropha eutropha), Ralstonia eutropha H16, Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodobacter sphaeroides ATCC 17025, Rhodopseudomonas palustris, Rhodopseudomonas palustris CGA009, Rhodopseudomonas palustris DX-1, Rhodospirillum rubrum, Rhodospirillum rubrum ATCC 11170, Ruminococcus obeum ATCC 29174, Saccharomyces cerevisiae, Saccharomyces cerevisiae cerevisiae)S288c, Salmonella enterica, Salmonella enterica subsp. Enterica serovar Typhimurium str. LT2, Salmonella enterica typhimurium, Salmonella enterica typhimuriumtyphimurium), Schizosaccharomyces pombe, Sebaldella termitidis ATCC 33386, Shewanella oneidensis MR-1, Sinorhizobium meliloti 1021, Streptomyces coelicolor, Streptomyces griseus subsp. Griseus NBRC 13350, Sulfolobus acidocalarius, Sulfolobus solfataricus P-2, Synechocystis str. PCC 6803, Syntrophobacter fumaroxidans, Thauera aromatica, Thermoanaerobacter sp.) X514, Thermococcus kodakaraensis, Thermococcus litoralis, Thermoplasma acidophilum, Thermoproteus neutrophilus, Thermotoga maritima, Thiocapsarose opersicina, Tolumonas auensis DSM 9187, Trichomonas vaginalis G3, Trypanosoma brucei, Tsukamurella paurometabola DSM 20162, Vibrio cholera, Vibrio harveyi ATCC BAA-1116, Xanthobacter autotrophicus Py2, Yersinia intermedia, or Zea mays. mays).

[0188] Genetic modification methods

[0189] Microbial cells can be engineered for fermentative production of progesterone and / or UDCA using conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry within the skill of the art. Such techniques are fully explained in the literature; see, for example, Sambrook et al., eds., Molecular Cloning: A Laboratory Manual, 4th ed., 2012; MJ Gait, ed., Oligonucleotide Synthesis, 1984); RI Freshney, ed., Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th ed., 2010; Academic Press, Inc., Current Protocols in Molecular Biology; FM Ausubel et al., eds., Current Protocols in Molecular Biology, 1987, and regularly updated; Mullis et al., eds., PCR: The Polymerase Chain Reaction, 1994; Singleton et al., eds., Dictionary of Microbiology and Molecular Biology, 1996. Biology” (Dictionary of Microbiology and Molecular Biology), 2nd edition, J. Wiley & Sons (New York, NY 1994).

[0190] Vector is a polynucleotide vector for introducing genetic material into a cell. The vector used for the methods described herein can be linear or circular. The vector can be integrated into the target genome of the host cell or replicate independently in the host cell. For many applications, an integration vector that produces a stable transformant is preferred. The vector can include, for example, an origin of replication, a multiple cloning site (MCS) and / or a selectable marker, etc. The expression vector generally includes an expression cassette containing regulatory elements that can promote the expression of a polynucleotide sequence (usually a coding sequence) in a specific host cell. Vectors include, but are not limited to, integration vectors, prokaryotic plasmids, episomes, viral vectors, cosmids, and artificial chromosomes.

[0191] Exemplary regulatory elements that can be used in expression cassettes include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences). Such regulatory elements are described in, for example, Goeddel, Gene Expression Technology: Methods In Enzymology, 185, Academic Press, San Diego, Calif. (1990).

[0192] In certain embodiments, the vector can be used to introduce a system capable of genome editing, such as a CRISPR system. See U.S. Patent Publication No. 2014 / 0068797, published on March 6, 2014; see also Jinek M. et al., “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, 337: 816-21, 2012. In the type II CRISPR-Cas9 system, Cas9 is a site-directed nuclease, i.e., an enzyme that is directed or can be directed to a specific target sequence and utilizes two different endonuclease domains (HNH and RuvC / RNase H-like domains) to cleave polynucleotides. Because Cas9 is directed to its cleavage site by RNA, Cas9 can be modified to cleave DNA at any desired site. Therefore, Cas9 is also called an “RNA-guided nuclease.” More specifically, Cas9 associates with one or more RNA molecules, at least a portion of which hybridizes to a specific sequence in a target polynucleotide, thereby guiding Cas9 to a specific polynucleotide target. Ran, FA et al. ("In vivo genome editing using Staphylococcusaureus Cas9", Nature, 520(7546): 186-91, 2015, Apr. 9); including all extended data) introduced the crRNA / tracrRNA sequences and secondary structures of 8 type II CRISPR-Cas9 systems. Cas9-like synthetic proteins are also known in the art (see U.S. Patent No. 2014-0315985, published on October 23, 2014).

[0193] The present invention relates to the invention and the method for the preparation of the polynucleotide sequence of the present invention.Carrier or other polynucleotide can be introduced into microbial cells by various standard methods, for example, transform, engage, electroporation, nuclear microinjection, transduction, transfection (for example, lipofection-mediated or DEAE-dextrin-mediated transfection or the transfection using recombinant phage virus), hatch together with calcium phosphate DNA precipitate, carry out high-speed bombardment with the micro-bullet of DNA bag, and protoplast fusion.Transformant can be selected by any method known in the art.The suitable method of selecting transformant is described in U.S. Patent Publication 2009 / 0203102, 2010 / 0048964 and 2010 / 0003716 and International Patent Publication WO 2009 / 076676, WO 2010 / 003007 and WO 2009 / 132220.

[0194] Modified microbial cells

[0195] Said method can be used for producing the microbial cell of transformation, and the microbial cell of described transformation produces, and in certain embodiments overproduces, progesterone, 4CA3O, 3-keto-LCA, LCA and / or UDCA.Compared with natural microbial cell (any microbial host cell as described herein), the microbial cell of transformation can have at least 1,2,3,4,5,6,7,8,9,10,20,30,40,50,60,70,80,90,100 or more kinds of genetic alterations, such as 30-100 kinds of changes.The microbial cell of the transformation described in the following embodiment has one, two or three kinds of genetic alterations, but those skilled in the art can design the microbial cell with additional changes according to the guidance proposed herein.In certain embodiments, compared with natural microbial cell, the microbial cell of described transformation has and is no more than 15,14,13,12,11,10,9,8,7,6,5 or 4 genetic alterations. In various embodiments, microbial cells engineered to produce progesterone and / or UDCA can have a number of genetic alterations falling within any of the following exemplary ranges: 1-10, 1-9, 1-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-7, 3-6, 3-5, 3-4, etc.

[0196] In certain embodiments, the engineered microbial cell expresses at least two heterologous genes encoding, for example, non-native side chain cleavage P450 enzymes and / or one or more non-native redox chaperone proteins. In various embodiments, the microbial cell can include and express, for example: (1) a single copy of each of the two genes, (2) two or more copies of one of the two genes, which can be the same or different, or (3) two or more copies of each of the two genes, wherein the copies of any one of the genes can be the same or different. The same is true for other heterologous genes that can be introduced into the engineered microbial cell.

[0197] Such modified host cells may include at least one additional genetic alteration that increases the flux of any pathway leading to the production of a direct precursor of progesterone and / or UDCA (e.g., 4CA3O, 3-keto-LCA, or LCA). As discussed above, this can be achieved by increasing the activity of upstream enzymes and / or reducing the consumption of progesterone and / or UDCA precursors.

[0198] The modified microbial cells may contain introduced genes that have native nucleotide sequences or that differ from native nucleotide sequences. For example, the native nucleotide sequences may be codon-optimized for expression in specific host cells. For example, codon optimization for a specific host can be performed based on the codon usage table at www.kazusa.or.jp / codon / . The amino acid sequence encoded by any of these introduced genes may be native or differ from native. In various embodiments, the amino acid sequence has at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% amino acid sequence identity to the native amino acid sequence.

[0199] Culture of modified microbial cells

[0200] Any of the microbial cells described herein can be cultured for, e.g., maintenance, growth, and / or production of progesterone, 4CA3O, 3-keto-LCA, LCA, and / or UDCA. In certain embodiments, the microbial cells (e.g., bacteria) described herein have a phytosterol uptake rate in culture that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 5000-fold, or 10000-fold greater than that of yeast producing progesterone, 4CA3O, 3-keto-LCA, LCA, and / or UDCA.

[0201] In certain embodiments, the culture is grown to an absorbance at 600 nm of 10-500, such as an absorbance of 50-150.

[0202] In various embodiments, the culture comprises progesterone, 4CAO, 3-keto-LCA, LCA and / or UDCA produced at a titer of at least 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, or 900 micrograms / liter, or at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150 mg / liter. In various embodiments, the titer is between 50 μg / L to 100 mg / L, 75 μg / L to 75 mg / L, 100 μg / L to 50 mg / L, 200 μg / L to 25 mg / L, 300 μg / L to 10 mg / L, 350 μg / L to 5 mg / L, or any range bounded by any of the foregoing values.

[0203] culture medium

[0204] Microbial cells can be cultured in any suitable medium, including but not limited to a minimal medium, i.e., a medium containing the minimum nutrients necessary for cell growth. Minimal media typically contain: (1) a carbon source for microbial growth; (2) salts, which may depend on the specific microbial cells and growth conditions; and (3) water. Suitable media may also include any combination of the following: a nitrogen source for growth and product formation, a sulfur source for growth, a phosphate source for growth, a metal salt for growth, vitamins for growth, and other cofactors for growth.

[0205] Any suitable carbon source can be used to culture host cells. The term "carbon source" refers to one or more carbon-containing compounds that can be metabolized by microbial cells. In various embodiments, the carbon source is a carbohydrate (e.g., monosaccharide, disaccharide, oligosaccharide or polysaccharide) or invert sugar (e.g., enzyme-treated sucrose syrup). Exemplary monosaccharides include glucose (dextrose), fructose (levulose) and galactose; exemplary oligosaccharides include dextran or glucan, and exemplary polysaccharides include starch and cellulose. Suitable sugars include C6 sugars (e.g., fructose, mannose, galactose or glucose) and C5 sugars (e.g., xylose or arabinose). Other cheaper carbon sources include sugarcane juice, beet juice, sorghum juice, etc., any of which can be completely or partially deionized, but this step is not required.

[0206] The salts in the culture medium generally provide essential elements, such as magnesium, nitrogen, phosphorus, and sulfur, to allow cells to synthesize proteins and nucleic acids.

[0207] Minimal culture media may be supplemented with one or more selective agents, such as antibiotics.

[0208] To produce progesterone, 4CA3O, 3-keto-LCA, LCA and / or UDCA, the culture medium may include and / or be supplemented during the culture period with glucose and / or a nitrogen source such as urea, ammonium salts, ammonia, or any combination thereof.

[0209] Culture conditions

[0210] The materials and methods suitable for microbial cell maintenance and growth are well known in the art. For example, refer to U.S. Patent Publication 2009 / 0203102, 2010 / 0003716 and 2010 / 0048964 and International Publication WO 2004 / 033646, WO 2009 / 076676, WO 2009 / 132220 and WO 2010 / 003007, the compilations such as Gerhardt, " Manual of Methods for General Bacteriology ", American Society for Microbiology (" Manual of General Bacteriology Methods ", American Society for Microbiology), Washington, DC (1994), or " Brock in Biotechnology ", Industrial Microbiology textbook, second edition (1989), Sinauer Associates, Inc., Sunderland, Mass.

[0211] In general, cells are grown and maintained at an appropriate temperature, gas mixture, and pH (e.g., about 20° C. to about 37° C., (about 6% to about 84% CO 2 , and a pH between about 5 and about 9). In some aspects, cells are grown at 28-34° C. (e.g., about 28° C., about 29° C., about 30° C., about 31° C., about 32° C., about 33° C., or about 34° C.). In certain embodiments, for example, when thermophilic bacteria are used as host cells, higher temperatures (e.g., 50° C.-75° C.) can be used. In some aspects, the pH range of the fermentation is between about pH 5.0 and about pH 9.0 (e.g., about pH 6.0 to about pH 8.0, or about 6.5 to about 7.0). Depending on the requirements of the particular cells, the cells can be grown aerobically, anaerobically, or anaerobicly.

[0212] Operable standard culture conditions and fermentation mode, for example batch, flow addition or continuous fermentation, are described in U.S. Patent Publication 20072013131, 2009 / 0203102, 2010 / 0003716 and 2010 / 0048964, and international publication WO2009 / 076676, WO2009 / 132220 and WO2010 / 003007.Batch and flow addition fermentation are common and well-known in the art, and related examples can be referring to " Brock in Biotechnology ", industrial microbiology textbook, second edition (1989), Sinauer Associates, Inc., Sunderland, Mass.

[0213] Production and recovery of progesterone, 4CA3O, 3-keto-LCA, LCA and / or UDCA

[0214] Any of the methods described herein may further include a step of recovering progesterone, 4CA3O, 3-keto-LCA, LCA, and / or UDCA. In certain embodiments, the progesterone, 4CA3O, 3-keto-LCA, LCA, and / or UDCA produced by the fermentation, contained in a so-called harvest stream, is recovered / harvested from the production vessel. The harvest stream may include, for example, a cell-free or cell-containing aqueous solution from the production vessel containing progesterone, 4CA3O, 3-keto-LCA, LCA, and / or UDCA produced by conversion of substrate by resting cells in the production vessel. The cells present in the harvest stream can be separated from the progesterone, 4CA3O, 3-keto-LCA, LCA, and / or UDCA by any procedure known in the art, such as filtration, centrifugation, decantation, cross-flow ultrafiltration or microfiltration, tangential flow ultrafiltration or microfiltration, or dead-end filtration. After such cell separation procedures, the harvest stream is substantially free of cells.

[0215] In a preferred embodiment, the progesterone, 4CA3O, 3-keto-LCA, LCA and / or UDCA produced can be further separated and / or purified from other components contained in the harvest stream, so-called downstream processing steps. These steps may include any means known to the skilled person, such as concentration, extraction, crystallization, precipitation, adsorption, ion exchange and / or chromatography. Any of these procedures can be used alone or in combination to purify progesterone, 4CA3O, 3-keto-LCA, LCA and / or UDCA. Further purification steps may include, for example, one or more of concentration, crystallization, precipitation, washing and drying, activated carbon treatment, ion exchange, nanofiltration and / or recrystallization. The design of a suitable purification scheme may depend on the cells, culture medium, volume of culture, production vessel, etc., and is within the level of those skilled in the art.

[0216] Example

[0217] The present invention is further described in the following examples, which are not intended to limit the scope of the invention described in the claims.

[0218] Example 1. Conversion of phytosterols to progesterone

[0219] Example 1.1 Preliminary Screening of Side-Chain Cleavage P450 Enzymes

[0220] Several side chain cleavage P450 enzymes and redox chaperones were selected for screening. Enzyme candidates were codon optimized for expression in Mycobacterium sp. Conventional methods (e.g., Gibson assembly) were used to clone the genes into Mycobacterium expression vectors. Each plasmid constructed contained a P450 gene paired with genes encoding ferredoxin (FDX_SPIOL_1) and ferredoxin reductase (FDXR_SPIOL_1) from spinach (Spinacia oleracea) with the chloroplast transit peptide removed. Each gene in the operon composed of the above genes was preceded by a ribosome binding site. Constitutive promoters Ptac or Ppms were used to control the expression of the operon. The expression vector backbone was derived from pJV53, a plasmid commonly used in recombination-mediated genetic engineering (recombinant engineering), containing a low-copy mycobacterium replication origin, an E. coli replication origin, and a kanamycin selectable marker. A total of six plasmids were constructed, as shown in Table 7.

[0221] Table 7. Plasmids used for preliminary screening of side-chain cleavage P450 genes

[0222] plasmids Plasmid description P001 Empty plasmid P002 Ppms>CYP_9SPHN_1>FDX_SPIOL_1>FDXR_SPIOL_1 P003 Ppms>CYP_9SPHN_2>FDX_SPIOL_1>FDXR_SPIOL_1 P004 Ppms>CYP_NOVAD_1>FDX_SPIOL_1>FDXR_SPIOL_1 P005 Ptac>CYP_9SPHN_1>FDX_SPIOL_1>FDXR_SPIOL_1 P006 Ptac>CYP_9SPHN_2>FDX_SPIOL_1>FDXR_SPIOL_1 P007 Ptac>CYP_NOVAD_2>FDX_SPIOL_1>FDXR_SPIOL_1

[0223] Each plasmid was transformed individually into Mycobacterium neoaurum NRRL B-3805 by electroporation using conventional methods. The cells were plated on solid agar medium containing kanamycin for selection and cultured at 30°C for several days. Six individual colonies of each transformation reaction were inoculated into the wells of a 96-well plate containing pre-seed culture medium. The 96-well plate was incubated in a shaker at 30°C for 3 days. The resulting culture was subcultured into a new 96-well plate containing fresh seed culture medium. The plate was incubated in a shaker at 30°C for one day. The culture was transferred to a new 96-well plate containing 2-hydroxypropyl-β-cyclodextrin and 2 mg of 70% β-sitosterol. The plate was incubated in a shaker at 30°C for 3 days. After incubation, ethyl acetate was added to the plate. The plate was vortexed and shaken for extraction. Next, the plate was centrifuged and the organic layer was transferred to a new 96-well round-bottom plate and further diluted before being injected onto a gas chromatograph for analysis.

[0224] According to gas chromatography analysis, some strains accumulated progesterone ( Figure 2 No progesterone was observed in the wild-type strain transformed with the empty expression vector (P001) as a control strain.

[0225] Example 1.2 Construction and screening of strains with deleted sterol degradation pathway genes

[0226] In order to reduce the level of unwanted by-products and increase the pathway flux towards progesterone, the key genes (Table 8) in the phytosterol degradation pathway were deleted using an integration plasmid that can not be bred in mycobacteria. In brief, genomic DNA from Mycobacterium neoaurum NRRL B-3805 was used as a template to amplify each 800 to 1200 bps of the target sequence upstream and downstream to be deleted. Conventional molecular biology protocols were then used to insert the upstream and downstream homologous sequences of amplification into pJQ200SK to generate an integration plasmid. The integration plasmid contained gentamicin selection marker and SacB counter-selection marker.

[0227] Table 8. Target genes for deletion

[0228]

[0229] For the deletion (deletion) of each gene, conventional methods are adopted, by electroporation, corresponding integration plasmid is transformed into new gold mycobacterium (Mycobacterium neoaurum) NRRL B-3805, and it is spread on the solid agar medium containing gentamicin, and is hatched at 30 ℃ for a few days.Single bacterium colony is inoculated in the fresh pre-seed medium in 96 well plates, and is cultivated in shaking table for three days at 30 ℃.In order to carry out counter-selection, culture is diluted with sterile water and is spread on the solid agar medium containing sucrose, and is hatched at 30 ℃ for a few days.Several bacterium colonies are inoculated in the fresh pre-seed medium in 96 well plates, and is cultivated at 800rpm for three days in shaking table at 30 ℃.Gained culture is used as PCR template, to determine whether they contain the required gene deletion without resistance marker.After counter-selection, further gene deletion can be constructed in bacterial strain according to program as described herein.Bacterial strain (table 9) with different gene deletion combinations has been constructed.

[0230] Table 9 Mutant strains with various gene knockout combinations

[0231]

[0232]

[0233] Strains S001-S006 were screened using the bioconversion assay described in Example 1.1. Titer measurements were performed on an Agilent RapidFire rather than a gas chromatograph. S006 accumulated the lowest levels of androstenedione ( Figure 3 ).

[0234] Example 1.3 Screening of side-chain cleavage P450s in sterol degradation pathway-deficient strains

[0235] The smo3 deletion mutant (KO_smo3) was transformed with several P450 replication plasmids listed in Table 7. The strains were screened using the biotransformation assay described in Example 1.1. Compared to the strains from the wild-type strain, the strains from the smo3 deletion mutant accumulated higher levels of progesterone and lower levels of androstenedione ( Figure 4 ).

[0236] Example 1.4 Enzyme diversity screening of side-chain cleavage P450 candidate enzymes

[0237] Using the bioinformatics software BLAST, an additional 18 side-chain cleavage P450 enzyme candidates were discovered through homology searches. These candidate enzyme genes were codon-optimized for expression in Mycobacterium and cloned into an expression vector containing the coding genes for the redox chaperones FDX_SPIOL_1 and FDXR_SPIOL_1, with the co-expression of the three genes controlled by the Ppms promoter. Each gene in the operon is preceded by a ribosome binding site. It is expected that the co-expression of the three genes will be able to convert phytosterols into progesterone. As described in Example 1.1, the plasmid was transformed into a Mycobacterium smo3 deletion mutant and screened in a 96-well plate assay. It was identified that the progesterone titer of the enzyme candidate CYP_XXXXX_1 was 1.1 times higher than that of the enzyme CYP_NOVAD_1 ( Figure 5 ).

[0238] Example 1.5 Enzyme diversity screening of redox chaperone proteins

[0239] Several redox chaperone protein candidates were selected. These candidate genes were codon-optimized for expression in Mycobacterium and cloned into an expression vector containing the P450 gene CYP_9SPHN_2, with expression controlled by the Ppms promoter. Some constructs contain both ferredoxin and ferredoxin reductase. Other constructs contain only ferredoxin. Each gene in the operon is preceded by a ribosome binding site. As described in Example 1.1, the plasmids were individually transformed into Mycobacterium smo3 deletion mutants and screened in a 96-well plate assay. Compared with FDX_SPIOL and FDXR_SPIOL, several redox chaperone protein combinations were able to further enhance progesterone production ( Figure 6 ).

[0240] Example 1.6 Modification and screening of cytochrome P450 fusion proteins

[0241] P450 fusion proteins were rationally designed using conventional methods and cloned into a Mycobacterium expression vector. The arrangement of P450 and redox chaperone proteins was different in different fusion proteins. Two connecting peptides were tested for fusing different protein components (Table 10). Only two of the three gene components were fused in each design (the three gene components were P450 genes, redox protein genes, and redox protein reductase genes). These genes were placed under the control of the promoter Ppms. Each gene in the operon is preceded by a ribosome binding site. As described in Example 1.1, the plasmid was transformed into the S007 strain and screened in a 96-well plate assay, and the product concentration was then measured on an Agilent RapidFire. The three fusion protein designs produced 1.4 times more progesterone than the original design in the 96-well plate assay. In the original design CYP_XXXXX_1>FDX_SPIOL_1>FDXR_SPIOL_1, P450, ferredoxin, and ferredoxin reductase were expressed as separate enzyme components ( Figure 7 ).

[0242] Table 10 Connector peptide sequences used for fusion protein design

[0243]

[0244] Example 2 Conversion of phytosterols to ursodeoxycholic acid

[0245] Example 2.1 Construction of a mutant strain producing 4-cholic acid-3-one

[0246] 4-Cholic acid-3-one (4CA3O) is an intermediate in the biosynthetic pathway for converting phytosterols to ursodeoxycholic acid (UDCA) proposed in this disclosure ( Figure 8A ). According to the plant sterol degradation pathway, the destruction of specific genes in sterol-degrading bacteria may lead to the accumulation of 4-cholic acid-3-one. The subsequent introduction of heterologous genes may convert this intermediate into ursodeoxycholic acid. The key genes in the plant sterol degradation pathway were deleted in Mycobacterium neoaurum NRRL B-3805 using an integration plasmid that cannot be reproduced in the strain (Table 11). In brief, genomic DNA was used as a template to PCR amplify 800 to 1200bp upstream and downstream of the target sequence to be deleted. The integration plasmid was then generated by inserting the upstream and downstream homologous sequences into pJQ200SK using conventional molecular biology protocols. The integration plasmid contains a gentamicin selection marker and a SacB counter-selection marker.

[0247] Table 11 Target genes deleted

[0248]

[0249] For each gene deletion, the corresponding integration plasmid was transformed into Mycobacterium neoaurum NRRL B-3805 or its derivative strain by electroporation using conventional methods and plated onto solid agar medium containing gentamicin and incubated at 30°C for several days. Single colonies were inoculated into fresh pre-seed medium containing gentamicin in 96-well plates and cultured in a shaker at 30°C for three days. For counter-selection, the culture was diluted with sterile water and plated onto solid agar medium containing sucrose and incubated at 30°C for four days. Several colonies were inoculated into fresh pre-seed medium in 96-well plates and cultured in a shaker at 30°C for three days. The resulting cultures were used as PCR templates to determine whether they contained the desired non-resistance marker gene deletion. After successful counter-selection, further gene deletions were constructed using new host strains according to the same procedure. The gene deletion targets are listed in Table 11. The strains produced using the above method are listed in Table 12.

[0250] Table 12 Strain description

[0251]

[0252]

[0253] Example 2.2 Production of 4-cholic acid-3-one by Mycobacterium knockout mutants

[0254] Four to six colonies of the strain produced by the method described in Embodiment 2.1 are inoculated into the wells of a 96-well plate containing fresh pre-seed medium together with appropriate control strains. The 96-well plate is incubated at 30°C in a shaker for 3 days. The resulting culture is subcultured into a new 96-well plate containing fresh seed medium. The plate is incubated at 30°C in a shaker for 1 day. The resulting culture is transferred to a new 96-well plate containing 2-hydroxypropyl-β-cyclodextrin and 2 mg of 70% β-sitosterol. The plate is incubated at 30°C in a shaker for 1-3 days. After incubation, ethyl acetate is added to the plate, vortexed and shaken for extraction. The plate is centrifuged, and the organic layer is transferred to a new 96-well round-bottom plate and further diluted, and then analyzed by liquid chromatography-mass spectrometry or gas chromatography on a triple quadruple instrument.

[0255] Accumulation of 4-cholic acid-3-one (4CA3O) was detected in several strains at a higher level than that of the wild-type strain S001 ( Figure 9 ). Compound accumulation was generally higher after 1 day compared to 3 days. For strain S007, the titer of 4-cholic acid-3-one was similar between the 1 day and 3 day time points.

[0256] Example 2.3 Overexpression of thioesterase

[0257] Overexpression of thioesterases can improve the hydrolysis of the pathway intermediate 3-oxo-chole-4-ene-24-acyl-CoA (3-OCO-CoA) to 4-cholic acid-3-one (4CA3O) and further increase the titer of the latter. Selected thioesterase candidates were found by homology search using BLAST. The thioesterase candidate genes were codon-optimized for expression in mycobacteria. At the same time, native thioesterases can also catalyze this reaction. The thioesterase in Mycobacterium neoaurum NRRL B-3805 was found by conventional bioinformatics methods. All thioesterases found were cloned into mycobacterium expression vectors and controlled by different constitutive and inducible promoters. Each plasmid was individually transformed into strain S007 by electroporation using conventional methods. The cells were plated onto solid agar medium containing kanamycin for selection and incubated for several days at 30° C. The resulting strains were screened using the 96-well plate assay described in Example 2.2.

[0258] Example 2.4 Construction of gene deletion to increase 4-cholic acid-3-one production

[0259] There may be other native enzymes that can reduce the accumulation of 4-cholic acid-3-one. For example, this product can be further activated by acyl-CoA ligase and the side chain degraded to form 3-oxo-choles-4-ene-24-acyl-CoA (3-OCO-CoA). 3-OCO-CoA can then be further metabolized to androstenedione and other byproducts ( Figure 8B ). Native acyl-CoA ligase candidates can be found by homology search. Alternatively, 3-OCO-CoA can be reduced to 24-hydroxychole-4-ene-3-one (24HC3) by two consecutive reductions under the catalysis of acyl-CoA reductase. Alternatively, the first reduction is catalyzed by acyl-CoA reductase and the second reduction is catalyzed by aldehyde reductase. Using known, characterized enzymes as the objects to be searched, candidates for the enzymes are found in the B3805 strain by homology search. An integration plasmid is designed to delete the genes for the above-mentioned candidate enzymes. In strains that produce 4-cholic acid-3-one (such as strain S007), these genes are deleted in different combinations. The resulting strains are transformed according to the method described in Example 2.2 and tested for their ability to produce 4-cholic acid-3-one. It is expected that gene deletion will increase the yield of 4-cholic acid-3-one.

[0260] Example 2.5 Gene deletion to eliminate by-product formation

[0261] To eliminate the production of byproducts androstenedione, androstenedione, and diol, other genes in the sterol degradation pathway can be deleted. Using the previously characterized and described enzyme from Mycobacterium tuberculosis H37Rv, a homology search was performed to identify genes in the sterol degradation pathway of Mycobacterium neoaurum NRRL B-3805 that are downstream of the intermediate 3-oxo-choles-4-ene-24-acyl-CoA) and upstream of the listed byproducts. Figure 8B As shown in Figure 2.1, these genes include kstD, chsH3, hsd4A, and ltp2. As described in Example 2.1, integration plasmids were designed to knock out these genes. The strain producing 4-cholic acid-3-one was transformed as described in Example 2.2, deleting these genes in different combinations. The knockout strain was tested for its ability to produce 4-cholic acid-3-one, androstenedione, androstenedione, and diolol as described in Example 2.2. It is expected that the gene deletion will reduce the content of the latter three compounds while maintaining or increasing the production of 4-cholic acid-3-one.

[0262] Example 2.6 Construction of gene deletion to prevent conversion of lithocholic acid and ursodeoxycholic acid

[0263] Lithocholic acid (LCA) is a potential precursor to ursodeoxycholic acid (UDCA) in the biosynthetic pathway proposed in this invention. The primary difference between these two molecules is the presence of a 7β-hydroxyl group in ursodeoxycholic acid. Native enzymes exist in mycobacteria that can act on both lithocholic acid and ursodeoxycholic acid and prevent their accumulation. Reactions catalyzed by these enzymes may include oxidation of the 3α-hydroxyl group and oxidation of the 5-carbon position to form a double bond between the 4 and 5 positions of LCA and UDCA. Other known possible reactions catalyzed by microorganisms include oxidation of the 7β-hydroxyl group of UDCA, epimerization, and dehydroxylation.

[0264] Various bioinformatics methods were used to identify candidate genes in Mycobacterium neoaurum NRRLB-3805 that may catalyze the above-mentioned reactions. Briefly, a number of enzymes with these or similar activities were selected, and these sequences were used as query sequences for BLAST searches or other homology-based searches to identify candidate genes in Mycobacterium neoaurum NRRL B-3805. Then, as described in Example 2.1, integration plasmids were designed to knock out these candidate genes. In the strain producing 4-cholic acid-3-one from plant sterols in Example 2.2, these genes were knocked out in different combinations. The resulting strains were screened in a 96-well plate assay as described in Example 2.2, but in which 70% of the β-sitosterol was replaced by LCA or UDCA. LCA and UDCA levels were measured. Strains that accumulated more LCA and UDCA than the parent strain may have reduced candidate enzyme activity due to gene deletion. These strains can serve as suitable parent strains for further strain engineering to produce LCA and UDCA.

[0265] Example 2.7 Production of lithocholic acid from phytosterols using Mycobacterium

[0266] The conversion of 4-cholic acid-3-one to lithocholic acid requires overexpression of 3α-hydroxysteroid dehydrogenase (3α-HSD) and 5β-steroid reductase (5βR). Candidate enzymes can be selected by homology search and codon optimized for expression in mycobacteria. The codon-optimized genes are cloned into expression vectors to co-express different combinations of 3α-HSD and 5βR under the control of the constitutive promoter Ppms. Each gene is cloned downstream of a ribosome binding site. The plasmid is transformed into a mycobacterium strain capable of producing 4-cholic acid-3-one from phytosterols, and the strain has reduced metabolic activity at the functional groups at the 3, 5, and 7 carbon positions due to the gene knockout described in Example 2.6. As described in Example 2.2, the strains were subjected to a well plate assay using phytosterols as substrates. It is expected that some strains will accumulate detectable levels of LCA.

[0267] Example 2.8 Production of Ursodeoxycholic Acid from Lithocholic Acid Using Mycobacterium

[0268] The conversion of LCA to UDCA requires overexpression of 7β-hydroxylase. Candidate enzymes can be selected by homology search and codon optimized for expression in mycobacteria. The codon-optimized gene is cloned into an expression vector so that 7β-hydroxylase and a suitable redox chaperone can be co-expressed. The redox chaperone can include ferredoxin and ferredoxin reductase, or cytochrome P450 reductase. Each gene is located downstream of a ribosome binding site, and the operon consisting of the genes is controlled by the promoter Ppms. The plasmid is transformed into a mycobacterium strain that produces 4-cholic acid-3-one from phytosterols and, due to the gene knockout described in Example 2.6, has reduced metabolic activity at the functional groups at the 3, 5, and 7 carbon positions. As described in Example 2.2, the strains were subjected to a well plate assay using lithocholic acid as a substrate. It is expected that some strains will accumulate detectable levels of UDCA.

[0269] Example 2.9 Production of Ursodeoxycholic Acid from Phytosterols Using Mycobacterium

[0270] The functional 7β-hydroxylase identified in Example 2.8 was cloned into expression vectors in various combinations with suitable redox chaperones and the functional 3α-HSD and 5βRs determined in Example 2.7. Each plasmid contained one copy of each of the genes, and each gene was located downstream of a ribosome binding site, and the operon composed of the genes was controlled by the promoter Ppms. The plasmid was transformed into a Mycobacterium strain that produces 4-cholic acid-3-one from phytosterols and, due to the gene knockout described in Example 2.6, reduced metabolic activity at the functional groups at the 3, 5, and 7 carbon positions. As described in Example 2.2, the strains were subjected to a well plate assay using phytosterols as substrates. It is expected that some strains will accumulate detectable levels of UDCA. The functional design was further optimized by adjusting the promoter strength, ribosome binding site, gene arrangement, and operon number.

[0271] Example 2.10 Gene deletion further increases 4-cholic acid-3-one production

[0272] To further enhance the conversion of phytosterols to 4-cholic acid-3-one (4CA3O), several additional candidate genes were disrupted based on the strategies described in Examples 2.1, 2.4, and 2.5. chsE4 and chsE5 from Mycobacterium tuberculosis together encode a heterotetrameric acyl-CoA dehydrogenase. Based on in vitro studies, it has been proposed that this enzyme complex catalyzes the dehydrogenation of the first acyl-CoA intermediate in the cholesterol degradation pathway. In theory, disruption of these two genes should eliminate the accumulation of 4CA3O. However, given the multi-substrate activity of the enzyme complex towards acyl-CoA intermediates with shorter side chain lengths, we sought to determine the effect of deleting these genes on 4CA3O production. It is possible that other, unknown, native enzymes exist that can catalyze the dehydrogenation of the first acyl-CoA intermediate with greater specificity. These enzymes would be able to compensate for the disruption of chsE4 and / or chsE5 and enable the accumulation of 4CA3O.

[0273] fadD17 encodes a putative acyl-CoA ligase that activates 4CA3O to 3-oxo-cholene-4-yl-24-acyl-CoA (3-OCO-CoA). 3-OCO-CoA is then further metabolized to androstenedione and other byproducts. The enzyme also acts on lithocholic acid and ursodeoxycholic acid to form the corresponding CoA esters, activating these compounds for degradation.

[0274] kstD encodes 3-ketosteroid-1-δ-dehydrogenase, which catalyzes the Δ1-dehydrogenation of steroids. This enzyme may have the activity to reduce the accumulation of 4CA3O and related compounds during fermentation.

[0275] Integration plasmids were designed to knock out the genes described in Example 2.1. In the strains producing 4-cholic acid-3-one, these genes were deleted in different combinations. The target genes for deletion are listed in Table 13. The resulting strains were constructed as described in Example 2.1 and their ability to convert phytosterols to other steroids was determined as described in Example 2.2. The constructed strains are listed in Table 14. Figure 12 As shown, all gene disruptions significantly increased the accumulation of 4-cholic acid-3-one relative to the initial starting strain S007. In addition, disruption of chsE4 and chsE5 with or without fadD17 significantly reduced the accumulation of androstenedione (S010 and S011).

[0276] Disruption of the above genes may reduce the degradation of ursodeoxycholic acid, which will be beneficial for the development of strains that produce UDCA. In the well plate assay, ursodeoxycholic acid was used instead of phytosterols to determine the ability of the constructed strain to degrade ursodeoxycholic acid. Figure 13As shown, disruption of chsE4, chsE5, and fadD17 in S011 resulted in decreased UDCA degradation and reduced AD accumulation compared with its parental and ancestral strains S008 and S007.

[0277] Table 13 Target genes deleted

[0278]

[0279] Table 14 Strain description

[0280]

[0281] Example 2.11 Identification of 5β-steroid reductase for the production of 3-keto-lithocholic acid from 4-cholic acid-3-one

[0282] The conversion of 4-cholic acid-3-one to 3-keto-lithocholic acid requires overexpression of 5β-steroid reductase (5βR). Candidate enzymes were selected by literature search and homology search and codon optimized for expression in Mycobacterium. The codon-optimized gene was cloned into an expression vector to express 5βR under the control of the Mycobacterium native promoter Psmo3. The plasmid was transformed into the Mycobacterium strain S007 that can produce 4-cholic acid-3-one from phytosterols. As described in Example 2.2, the strains were subjected to a well plate assay using phytosterols as substrates. Based on the results of liquid chromatography-mass spectrometry (LC-MS), several strains produced 3-ketolithocholic acid. As Figure 14 As shown, the 3-keto-lithocholic acid signal was significantly above the background level of the negative control strain not expressing 5βR.

[0283] Example 2.12 Production of lithocholic acid from phytosterols using Mycobacterium

[0284] The conversion of 4-cholic acid-3-one to lithocholic acid requires overexpression of 3α-hydroxysteroid dehydrogenase (3α-HSD) and 5β-steroid reductase (5βR). Candidate 3α-HSD enzymes were selected through literature search and homology search and codon optimized for expression in Mycobacterium. The codon-optimized genes were cloned into expression vectors to co-express different combinations of 3α-HSD and 5BR_ARATH_1 under the control of promoter Psmo3. Each gene was cloned downstream of a ribosomal binding site. The plasmids were transformed into Mycobacterium strain S007, which can produce 4-cholic acid-3-one from phytosterols. The strains were tested in a well plate assay using phytosterols as substrates as described in Example 2.2. As Figure 15 As shown, several strains accumulated detectable levels of lithocholic acid (LCA), which far exceeded the background levels of negative control strains that do not express these two enzymes.

[0285] Example 2.13 Production of Ursodeoxycholic Acid from Lithocholic Acid Using Mycobacterium

[0286] The conversion of LCA to UDCA requires overexpression of 7β-hydroxylase. Candidate enzymes were selected through literature and homology searches and codon-optimized for expression in Mycobacterium. The codon-optimized genes were cloned into expression vectors capable of co-expressing 7β-hydroxylase and redox chaperones: ferredoxin (FDX_SYNY4_2; SEQ ID NO: 137) and ferredoxin reductase (FDXR_SYNY4_2; SEQ ID NO: 136) from Synechocystis sp. The ferredoxin reductase contained an N-terminal truncation to remove a predicted transit peptide. Each gene is located downstream of a ribosome binding site, and the operon comprising the genes is controlled by the promoter Psmo3. The plasmids were transformed into Mycobacterium strain S007, which is capable of producing 4-cholic acid-3-one from phytosterols. The strains were subjected to plate assays as described in Example 2.2, but using 1 g / L lithocholic acid instead of phytosterols as substrate. Figure 16 As shown, several strains accumulated UDCA to detectable levels, exceeding the background level of the negative control.

[0287] Example 2.14 Production of Ursodeoxycholic Acid from Phytosterols Using Mycobacterium

[0288] The functional 7β-hydroxylase identified in Example 2.13 was cloned into an expression vector together with the redox chaperones FDX_SYNY4_2 and FDXR_SYNY4_2 and the functional 3α-HSD and 5βR identified in Examples 2.11 and 2.12. Each plasmid contains one copy of each of the genes, and each gene is located downstream of a ribosome binding site. The 7β-hydroxylase and redox chaperone are placed in one operon, and the 3α-HSD and 5βR are placed in another operon. Several promoters, including Pg13, Psmo3 and PkshA, are used to control the transcription of each operon. The plasmids were transformed into the Mycobacterium strain S007 that can produce 4-cholic acid-3-one from phytosterols. The plasmid description is listed in Table 15. As described in Example 2.2, the strains were subjected to a well plate test using phytosterols as substrates. As Figure 17 As shown, several strains produced UDCA to detectable levels, far exceeding control strains that did not express either of these enzymes.

[0289] Table 15 Plasmids designed for conversion of phytosterols to UDCA

[0290]

[0291] Other implementations

[0292] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A modified microbial cell that produces progesterone, wherein the modified microbial cell expresses: (a) non-native side-chain cleavage P450 enzymes; and (b) one or more non-native redox chaperone proteins; wherein, The one or more non-native redox chaperone proteins comprise a non-native ferredoxin and a non-native ferredoxin reductase; Wherein, the non-native side chain cleavage P450 enzyme is a Novosphingobium sp. PP1Y side chain cleavage P450 enzyme; in, (a) the non-native ferredoxin is a Synechococcus elongatus ferredoxin; and the non-native ferredoxin reductase is a Synechococcus longatus ferredoxin reductase; (b) the non-native ferredoxin is a Chlamydomonas reinhardtii ferredoxin; the non-native ferredoxin reductase is a Synechococcus longatus ferredoxin reductase; or (c) the non-native ferredoxin is spinach (Spinacia oleracea) ferredoxin; and the non-native ferredoxin reductase is spinach (Spinacia oleracea) ferredoxin reductase; Furthermore, the smo3 expression activity of the modified microbial cells is reduced or knocked out.

2. The modified microbial cell of claim 1 , wherein the modified microbial cell comprises an expression vector comprising: a first gene sequence encoding the non-native side-chain cleavage P450 enzyme, and a second gene sequence encoding the non-native ferredoxin and a non-native ferredoxin reductase.

3. The engineered microbial cell of claim 1 , wherein the engineered microbial cell comprises an expression vector comprising: a first gene sequence encoding the non-native side-chain cleavage P450 enzyme, a second gene sequence encoding a non-native ferredoxin, and a third gene sequence encoding a non-native ferredoxin reductase.

4. The modified microbial cell of claim 2 or 3, wherein the expression vector further comprises a promoter.

5. The modified microbial cell of claim 4, wherein the promoter is a constitutive promoter.

6. The modified microbial cell of claim 4, wherein the promoter is Ptac or Ppms.

7. The modified microbial cell of any one of claims 2-6, wherein the expression vector further comprises a ribosome binding site upstream of the first gene sequence, the second gene sequence, and the third gene sequence.

8. The engineered microbial cell of any one of claims 2-7, wherein all three of the first gene sequence, the second gene sequence, and the third gene sequence are linked to express a fusion protein.

9. The modified microbial cell of claim 8, wherein the fusion protein comprises a connecting peptide sequence.

10. The modified microbial cell of any one of claims 2-9, wherein the expression vector comprises from the 5' end to the 3' end: (a) a second gene sequence encoding a spinach (Spinacia oleracea) ferredoxin protein, (b) a first gene sequence encoding a Novosphingobium sp. PP1Y side-chain cleavage P450 enzyme, and (c) a third gene sequence encoding a spinach (Spinacia oleracea) ferredoxin reductase; The spinach (Spinacia oleracea) ferredoxin and the Novosphingobium sp. PP1Y side chain cleavage P450 enzyme are connected to form a fusion protein via a connecting peptide.

11. The modified microbial cell of any one of claims 2 to 9, wherein the expression vector comprises from the 5' end to the 3' end: (a) a first gene sequence encoding a Novosphingobium sp. PP1Y side-chain cleavage P450 enzyme, (b) a second gene sequence encoding a spinach (Spinacia oleracea) ferredoxin protein, and (c) The third gene sequence encoding the spinach (Spinacia oleracea) ferredoxin reductase.

12. The modified microbial cell of any one of claims 2 to 9, wherein the expression vector comprises, from the 5' end to the 3' end, (a) a first gene sequence encoding a Novosphingobium sp. PP1Y side-chain cleavage P450 enzyme, (b) a second gene sequence encoding a spinach (Spinacia oleracea) ferredoxin protein, and (c) a third gene sequence encoding a spinach (Spinacia oleracea) ferredoxin reductase; The Novosphingobium sp. PP1Y side chain cleavage P450 enzyme and the Spinacia oleracea ferredoxin are connected to form a fusion protein via a connecting peptide.

13. The engineered microbial cell of claim 1 , wherein the engineered microbial cell comprises one or more gene knockouts comprising: (a)smo3; (b) cyp142, smo3, and smo1; (c) cyp142, smo3, smo2, and kstD; (d) cyp142, smo3, smo2, chsH3, and kstD; (e) cyp142, smo3, smo2, and smo1; or (f) cyp142, smo3, smo2, smo1, and smo4.

14. A method for culturing the modified microbial cell of any one of claims 1 to 13, the method comprising: The cells are cultured under conditions suitable for the production of progesterone.

15. The method of producing a modified microbial cell according to claim 14, further comprising recovering the progesterone from the culture.

16. A method of producing a modified microbial cell according to claim 14, wherein the cell is cultured in the presence of phytosterols.

17. A method for transforming microbial cells according to claim 16, wherein the phytosterol is β-sitosterol, β-sitostanol, campesterol, stigmasterol and / or brassicasterol.

18. A culture of modified microbial cells comprising the modified microbial cells of any one of claims 1-17.

19. The culture of claim 18, wherein the culture comprises an amount greater than 0.01 mg / L, 0.05 mg / L, 0.1 mg / L, 0.5 mg / L, 1 mg / L, 5 mg / L, 10 mg / L, 50 mg / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L , 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, 55 g / L, 60 g / L, 65 g / L, 70 g / L, 75 g / L, 80 g / L, 85 g / L, 90 g / L, 95 g / L or 100 g / L of progesterone, 4CA3O, 3-keto-LCA, LCA and / or UDCA.

20. A method for producing pregnenolone and / or progesterone, the method comprising: Contacting the engineered microbial cell of any one of claims 1 to 17 with a sterol, thereby producing pregnenolone and / or progesterone.

21. A method for producing pregnenolone and / or progesterone according to claim 20, wherein the sterol is a phytosterol.

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