Methods for hydroxylating steroids

Abstract

The present invention relates to a 7-deoxysteroid having the general formula (I) at the 7-position. JPEG2023520307000023.jpg61108 a steroid having the general formula (II) JPEG2023520307000024.jpg61110 The present invention relates to an enzyme and method for hydroxylating hydroxylated amino acids.

Description

[Technical Field]

[0001] The present invention relates to means and methods for the hydroxylation of steroids. [Background technology]

[0002] 3,7,12-Trihydroxylated bile acids, such as cholic acid (3α,7α,12α-trihydroxy-5β-cholanic acid) or ursocholic acid (3α,7β,12α-trihydroxy-5β-cholanic acid), are industrially important chemicals, particularly as starting materials for the production of ursodeoxycholic acid (UDCA), which is used as a pharmaceutical agent, inter alia, to dissolve minor x-ray-negative gallstones and to treat the liver diseases primary ciliary cirrhosis and primary sclerosing cholangitis.

[0003] The most important industrial source of 3,7,12-trihydroxylated bile acids is bile juice from gallbladders, which accumulate as slaughterhouse waste in meat production. In addition to other animal species, bovine bile is often used. There is no industrially relevant total synthesis of 3,7,12-trihydroxylated bile acids. Because bile acid production is linked to another product (meat), the response to increased demand can be very limited. For this reason, it is of great interest to use raw bile as efficiently as possible.

[0004] Because bile is an aqueous mixture of bile acids, lipids, cholesterol, and other substances, component separation during bile acid extraction is particularly important. Bile acids also comprise a mixture whose components differ in the number and position of hydroxyl groups. In addition to cholic acid, bovine bile also contains a significant proportion of deoxycholic acid, which differs from cholic acid in that it lacks an OH group at the 7-position (3α,12α-dihydroxy-5β-cholanic acid). Deoxycholic acid has much lower commercial value than 3,7,12-trihydroxylated bile acids. Therefore, there is industrial interest in converting deoxycholic acid to 3,7,12-trihydroxylated bile acids by selectively introducing a hydroxyl group at the 7-position.

[0005] During hydroxylation, an oxygen atom is formally introduced into a (non-activated) C-H bond in an oxidation reaction. In organic chemistry, these are very difficult reactions to perform. The OH group is often introduced through a roundabout route, for example, by adding water to a C=C double bond. Selective hydroxylation at specific positions in complex molecules (e.g., bile acids) is problematic because several chemically (almost) equivalent C-H bonds exist.

[0006] Certain actinobacteria and filamentous fungi are known to be able to catalyze the regio- and stereospecific hydroxylation of lithocholic acid (LCA) at the 7β position to ursodeoxycholic acid (UDCA) (Kollerov et al., Steroids, 78(3):370-378 (2013); Tonin et al. (Beilstein Journal of Organic Chemistry, 14:470-483 (2018)).

[0007] In addition, hydroxylation of deoxycholic acid at the 7-position by filamentous fungi (Kollerov et al., Steroids, 107:20-29 (2016)) and actinomycetes (Deshcherevskaya et al., Journal of Molecular Catalysis B: Enzymatic, 133:p.157-p.165 (2016)) has been reported.

[0008] It is an object of the present invention to provide means and methods for hydroxylating steroids, such as bile acids and their derivatives, particularly at this point, which have a hydrogen at the 7-position and no hydroxyl group. Summary of the Invention [Means for solving the problem]

[0009] The object of the present invention is to provide 7-deoxysteroids having in the 7-position the general formula (I) [ka] a steroid having the general formula (II) [ka] This is achieved by using a cytochrome P450 or a functional fragment thereof to hydroxylate During the ceremony, X1 and X2 are independently H, Cl, F, Br, I, CF3, a C1-C6 alkyl group, OH, a C1-C6 alkoxy group, CN, NO2, N(R6)2, an epoxy group, a CHO or a CO2R6 group, R6 is -C(O)H, -C(O)CH3, -C(O)CH2CH3, -C(O)(CH2)2CH3, -C(O)CH(CH3)2, -C(O)(CH2)3CH 3, -C(O)CH(CH3)CH2CH3, -C(O)CH2CH2(CH3)2, -C(O)C(CH3)3, -C(O)Ph, -C(O)CH2Ph, R1 and R2 are independently H, OH, OR7, or O, wherein: R7 is -C(O)H, -C(O)CH3, -C(O)CH2CH3, -C(O)(CH2)2CH3, -C(O)CH(CH3)2, -C(O)(CH2)3CH 3, -C(O)CH(CH3)CH2CH3, -C(O)CH2CH2(CH3)2, -C(O)C(CH3)3, -C(O)Ph, -C(O)CH2Ph, R3 is H, OH, OR8, C1~C 10 Alkyl groups, C1-C 10 an alkenyl group, —CHO, —C(O)(CH), —C(O)(CHOH), —CH(CH)C(O)CH, —CH(CH)((CH)COR) or —CH(CH)((CH)CONHR), wherein R8 is -C(O)H, -C(O)CH3, -C(O)CH2CH3, -C(O)(CH2)2CH3, -C(O)CH(CH3)2, -C(O)(CH2)3CH 3, -C(O)CH(CH3)CH2CH3, -C(O)CH2CH2(CH3)2, -C(O)C(CH3)3, -C(O)Ph or -C(O)CH2Ph, R9 is -CH3, -CH2COOH, -CH2CH3, -CH(CH3)2, -(CH2)2CH3, -(CH2)2SO3H, C(CH3)3, -(CH2)3CH3, -CH(CH3)CH2CH3, -CH2CH2(CH3)2, an aryl group or an alkylaryl group; R4 is H, OH or -OR 10 where: R 10 is -C(O)H, -C(O)CH3, -C(O)CH2CH3, -C(O)(CH2)2CH3, -C(O)CH(CH3)2, -C(O)(CH2)3CH3 , -C(O)CH(CH3)CH2CH3, -C(O)CH2CH2(CH3)2, -C(O)C(CH3)3, -C(O)Ph or -C(O)CH2Ph, R5 is H, CF3, a C1-C6 alkyl group, a C1-C6 alkenyl group, OH, O, or a C1-C6 alkoxy group, and the dashed line represents an optional double bond, provided that when ring A has a C4-C5 double bond, ring B does not have a double bond, and when X1 and X2 form an epoxy group, ring C does not have a double bond; The cytochrome P450 enzyme is characterized in that it comprises an amino acid sequence which is at least 80%, preferably at least 90%, in particular 100% identical to the amino acid sequence of SEQ ID NO: 1 or 2.

[0010] Surprisingly, it has been shown that cytochrome P450 and functional fragments thereof are capable of hydroxylating steroids such as cholic acid and its derivatives, respectively, having formula (I) at the 7-position.

[0011] A further aspect of the present invention relates to a method for preparing a steroid having general formula (II) as defined above, preferably cholic acid, or a derivative thereof, which method comprises converting a 7-deoxysteroid having general formula (I), preferably 7-deoxycholic acid, or a derivative thereof, with a cytochrome P450 or a functional mutant thereof according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0012] Cytochrome P450 and its functional mutants are surprisingly capable of selectively hydroxylating 7-deoxysteroids, such as 7-deoxycholic acid, and its derivatives at the 7-position.

[0013] According to the invention, the cytochrome P450 enzyme comprises an amino acid sequence that is at least 80%, preferably at least 90%, in particular 100% identical to the amino acid sequence of SEQ ID NO: 1 or 2.

[0014] Cytochromes P450 catalyze the monooxygenase reactions of numerous endogenous and exogenous substrates. They are involved in the metabolism of exogenous substrates such as steroids, eicosanoids, fatty acids and bile acids, as well as drugs, pesticides and chemical carcinogens, among others.

[0015] The cytochrome P450 according to the invention can be used from bacteria, such as, for example, Actinobacteria, in particular from the genus Streptomyces, for example, where the sequence can be isolated, for example, from a genomic DNA or cDNA library using known techniques.

[0016] The cytochrome P450s and their functional variants according to the present invention may be present in or isolated from their original organism, or they may be recombinantly expressed or synthetically produced. Recombinantly expressed polypeptides are preferably used according to the present invention.

[0017] Various established microorganisms can be used for the recombinant expression of the enzymes according to the invention, such as, for example, Escherichia coli (E. coli), Bacillus subtilis, Saccharomyces cerevisiae or Pichia pastoris. Suitable protocols in this regard are described in detail in the relevant specialist literature or are known to those skilled in the art.

[0018] According to the present invention, the enzymes / polypeptides are preferably used as recombinantly overexpressed proteins in E. coli, and the corresponding cell lysates are preferably used without further processing / purification or after relatively simple processing steps (e.g., centrifugation, precipitation, concentration, or lyophilization). After recombinant overexpression of the enzymes used, E. coli cells can alternatively be used directly in the reaction without cell disruption or, for example, after a freeze / thaw cycle. Suitable expression plasmids are known to those skilled in the art and can often be purchased commercially.

[0019] A "functional variant" of a cytochrome P450 may be a fragment or a variant of a cytochrome P450, where a fragment of a cytochrome P450 may also be referred to as a "functional fragment." A "functional variant" of a cytochrome P450 can catalyze the same reaction as the protein from which the variant is derived. Whether a variant is functional, i.e., catalyzes the same reaction as the protein from which the variant is derived, can be determined by establishing that the variant catalyzes the same reaction. For this purpose, the prior art or the methods described herein have been established, respectively. The conversion rate of a substrate by a functional variant according to the present invention may deviate from the conversion rate of the cytochrome P450 from which the variant is derived.

[0020] "Derivatives of 7-deoxysteroids" include compounds derived from 7-deoxysteroids and having a wide variety of modifications, with one or more modifications at positions 3, 12 and 17 of the 7-deoxysteroids being particularly preferred. Such modifications preferably include substitutions as defined above.

[0021] According to a preferred embodiment of the present invention, X1, X2, R4 and R5 are H; R1 and R2 are independently H, OH, OR8, or O, wherein: R8 is -C(O)H, -C(O)CH3, -C(O)CH2CH3, -C(O)(CH2)2CH3, -C(O)CH(CH3)2, -C(O)(CH2)3CH 3, -C(O)CH(CH3)CH2CH3, -C(O)CH2CH2(CH3)2, -C(O)C(CH3)3, -C(O)Ph, -C(O)CH2Ph, R3 is C1~C 10 Alkyl groups, C1-C 10 an alkylene group, —CH(CH3)((CH2)2CO2R9) or —CH(CH3)((CH2)2CONHR9), wherein R9 is -CH3, -CH2COOH, -CH2CH3, -CH(CH3)2, -(CH2)2CH3, -(CH2)2SO3H, C(CH3)3, -(CH2)3CH3, -CH(CH3)CH2CH3, -CH2CH2(CH3)2, an aryl group or an alkylaryl group.

[0022] According to a further preferred embodiment of the present invention, the aryl group is selected from the group consisting of a phenyl group, a phenyl group substituted with F, Cl, Br, NO2 or CH3, and heteroaryl.

[0023] According to yet another preferred embodiment of the present invention, the alkylaryl group is selected from the group consisting of a benzyl group, a halogenated benzyl group in which the halogen is F, Cl or Br, and a benzyl group substituted with NO2.

[0024] According to a preferred embodiment of the present invention, R1 is OH, R2 is O or OH, R3 is CH(CH3)((CH2)2CO2R5), R4 is H and R5 is H.

[0025] According to another preferred embodiment of the present invention, the 7-deoxysteroid having the general formula (II) is selected from the group consisting of 3α,12α-dihydroxy-5β-cholan-24-oic acid, 3α,12β-dihydroxy-5β-cholan-24-oic acid, 3β,12α-dihydroxy-5β-cholan-24-oic acid, 3β,12β-dihydroxy-5β-cholan-24-oic acid, 3β-hydroxy-12-keto-5β-cholan-24-oic acid, 3-keto,12β-hydroxy-5β-cholan-24-oic acid, 3-keto,12α-hydroxy-5β-cholan-24-oic acid, 3α-hydroxy-5β-cholan-24-oic acid, 3-keto-5β-cholan-24-oic acid, 3β-hydroxy-5β-cholan-24-oic acid and the esters of the respective acids.

[0026] The cytochrome P450 hydroxylases used according to the invention for the hydroxylation of 7-deoxysteroids and derivatives thereof having the general formula (I) to steroids or derivatives thereof having the general formula (II) comprise an amino acid sequence which is at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and especially 100% identical to the amino acid sequence of SEQ ID NO: 1 or 2. SEQ ID NO:1: [ka] SEQ ID NO:2: [ka]

[0027] The amino acid sequences of SEQ ID NOs: 1 and 2 are preferably encoded by the nucleic acid sequences of SEQ ID NOs: 3 and 4, and the nucleic acid sequences of SEQ ID NOs: 5 and 6 are optimized for expression in E. coli. SEQ ID NO:3: [ka] SEQ ID NO:4: [ka] SEQ ID NO:5: [ka] SEQ ID NO:6: [ka]

[0028] As used herein, "identical" means that two or more amino acid sequences, when superimposed, can have a certain degree of "identity" with each other (matching amino acid residues at the same positions). "Identity" is defined herein as the percentage of amino acids in a qualifying amino acid sequence that are identical to those in a starting sequence, i.e., after alignment of two sequences and introduction of gaps, if necessary, to achieve the maximum percent sequence identity generated by the "Protein BLAST" program (blastp, Altschul et al., J. Mol. Biol. (1997) 215:403-410, http: / / blast.ncbi.nlm.nih.gov / Blast.cgi, commonly referred to herein as "BLAST"), with all variable parameters set to default values. Here, the algorithm "blastp (Protein-Protein-BLAST)" was used with the following parameters: "expect threshold": 0.05, "word size": 6, matrix: BLOSUM62, "gap cost": "Existence" 11, "Extension" 1, conditional composite score matrix adjustment, no filter, and no mask. The percent (%) value of amino acid sequence identity is determined by dividing the number of matching identical nucleotides by the sequence length for which the identity is recorded in percent.

[0029] A further aspect of the present invention relates to a process for preparing a steroid or a derivative thereof having the general formula (II): [ka] During the ceremony, X1 and X2 are independently H, Cl, F, Br, I, CF3, a C1-C6 alkyl group, OH, a C1-C6 alkoxy group, CN, NO2, N(R6)2, an epoxy group, a CHO or a CO2R6 group, R6 is -C(O)H, -C(O)CH3, -C(O)CH2CH3, -C(O)(CH2)2CH3, -C(O)CH(CH3)2, -C(O)(CH2)3CH 3, -C(O)CH(CH3)CH2CH3, -C(O)CH2CH2(CH3)2, -C(O)C(CH3)3, -C(O)Ph, -C(O)CH2Ph, R1 and R2 are independently H, OH, OR7, or O, wherein: R7 is -C(O)H, -C(O)CH3, -C(O)CH2CH3, -C(O)(CH2)2CH3, -C(O)CH(CH3)2, -C(O)(CH2)3CH 3, -C(O)CH(CH3)CH2CH3, -C(O)CH2CH2(CH3)2, -C(O)C(CH3)3, -C(O)Ph, -C(O)CH2Ph, R3 is H, OH, OR8, C1~C 10 Alkyl groups, C1-C 10 an alkenyl group, —CHO, —C(O)(CH), —C(O)(CHOH), —CH(CH)C(O)CH, —CH(CH)((CH)COR) or —CH(CH)((CH)CONHR), wherein R8 is -C(O)H, -C(O)CH3, -C(O)CH2CH3, -C(O)(CH2)2CH3, -C(O)CH(CH3)2, -C(O)(CH2)3CH 3, -C(O)CH(CH3)CH2CH3, -C(O)CH2CH2(CH3)2, -C(O)C(CH3)3, -C(O)Ph or -C(O)CH2Ph, R9 is -CH3, -CH2COOH, -CH2CH3, -CH(CH3)2, -(CH2)2CH3, -(CH2)2SO3H, C(CH3)3, -(CH2)3CH3, -CH(CH3)CH2CH3, -CH2CH2(CH3)2, an aryl group or an alkylaryl group; R4 is H, OH or -OR 10 where: R 10 is -C(O)H, -C(O)CH3, -C(O)CH2CH3, -C(O)(CH2)2CH3, -C(O)CH(CH3)2, -C(O)(CH2)3CH3 , -C(O)CH(CH3)CH2CH3, -C(O)CH2CH2(CH3)2, -C(O)C(CH3)3, -C(O)Ph or -C(O)CH2Ph, R5 is H, CF3, a C1-C6 alkyl group, a C1-C6 alkenyl group, OH, O, or a C1-C6 alkoxy group, and the dashed line represents an optional double bond, provided that when ring A has a C4-C5 double bond, ring B does not have a double bond, and when X1 and X2 form an epoxy group, ring C does not have a double bond; 7-deoxysteroids or derivatives thereof having the general formula (I) [ka] The method comprises a step of converting the cytochrome P450 enzyme or a functional variant thereof, wherein the cytochrome P450 enzyme comprises an amino acid sequence that is at least 80%, preferably at least 90%, particularly 100% identical to the amino acid sequence of SEQ ID NO: 1 or 2.

[0030] Using the method according to the invention, a 7-deoxysteroid having the general formula (I) or a respective derivative thereof can be converted into a steroid having the general formula (II) or a respective derivative thereof using a cytochrome P450 according to the invention or a functional variant thereof.

[0031] To support the redox reaction of the cytochrome P450 of the present invention or its functional variant, it is advantageous to carry out the method of the present invention in the presence of a reducing agent. NAD(P)H, flavin, or ferredoxin can be used as the reducing agent. For example, when using the redox cofactors NAD(P)+ and / or NAD(P)H, it is advantageous to use them at a concentration of 0.001 mM to 10 mM, more preferably 0.05 mM to 1 mM, in the reaction mixture.

[0032] The method according to the present invention is preferably carried out in the presence of a redox partner of cytochrome P450. Redox partners are understood to be proteins of the ferredoxin and ferredoxin reductase classes that are beneficial to the function of cytochrome P450 according to the present invention. Possible pairs of redox partners preferably include putidaredoxin and putidaredoxin reductase from Pseudomonas putida. Furthermore, those skilled in the art can identify additional ferredoxin proteins and ferredoxin reductases that are potential redox partners of cytochrome P450 according to the present invention. Their suitability as redox partners can be verified by functional assays, for example, as described in Examples 3 to 5. The putidaredoxins and / or putidaredoxin reductases used in these examples can be replaced by possible alternative proteins or enzymes, respectively. If sufficient formation of the desired product (eg, ursocholic acid) is observed, the tested redox partner can be considered a functional substitute for putidaredoxin and / or putidaredoxin reductase.

[0033] According to a particularly preferred embodiment of the invention, the ferredoxin used in the method according to the invention comprises an amino acid sequence which is at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, especially 100% identical to the amino acid sequence of SEQ ID NO: 7, wherein X is a methionine residue or is not an amino acid. SEQ ID NO:7: [ka]

[0034] According to a further preferred embodiment of the invention, the ferredoxin reductase used in the method according to the invention comprises an amino acid sequence which is at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and especially 100% identical to the amino acid sequence of SEQ ID NO: 8. SEQ ID NO:8: [ka]

[0035] The amino acid sequences of SEQ ID NOs: 7 and 8 are preferably encoded by the nucleic acid sequences of SEQ ID NOs: 9 and 10, respectively, and the nucleic acid sequences of SEQ ID NOs: 11 and 12 are optimized for expression in E. coli. SEQ ID NO:9: (ATG) 0又は1 [ka] SEQ ID NO:10: [ka] SEQ ID NO:11: (ATG) 0又は1 [ka] SEQ ID NO:12: [ka]

[0036] The expression of cytochrome P450 and ferredoxin and ferredoxin reductase according to the invention in bacteria, in particular E. coli, is particularly advantageous when nucleic acids having the nucleic acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11 and / or SEQ ID NO: 12 are used. Thus, a further aspect of the present invention relates to nucleic acids (DNA and / or RNA) having a nucleic acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11 and SEQ ID NO: 12, as well as vectors and / or cells, in particular E. coli cells, comprising at least one of these sequences.

[0037] It has been shown to be particularly advantageous when the above-mentioned ferredoxin and ferredoxin reductase are co-expressed with cytochrome P450 in a production strain (e.g., an E. coli strain). Co-expression of the three proteins or respective enzymes, ideally under the same promoter, allows for an ideal balance between the enzymes to be established, which has a particularly advantageous effect on the enzymatic conversion of the substrate.

[0038] According to a preferred embodiment of the present invention, the method according to the present invention is carried out at a temperature of 10° C. to 40° C., preferably 15° C. to 38° C., more preferably 20° C. to 30° C., more preferably 22° C. to 26° C. According to the present invention, it has been shown that the enzymatic activity of cytochrome P450 for the reaction according to the present invention is particularly high in this range.

[0039] According to a further preferred embodiment of the invention, the process according to the invention is carried out at a pH between 6.5 and 8.5, preferably between 7 and 8, more preferably between 7.2 and 7.8, at which pH values ​​the enzymatic activity of cytochrome P450 is highest to allow an adequate conversion of the substrate.

[0040] Hydroxylation of deoxysteroids or the respective deoxysteroid derivatives can be carried out regioselectively at position 7 of the steroid skeleton. In this way, in particular, the 7β-hydroxyl group can be introduced stereoselectively, so that, for example, ursocholic acid and / or ursocholic acid derivatives can be produced.

[0041] In the process according to the present invention, the isolation of the product can be carried out in different ways. For example, the product can be extracted from the reaction mixture with a suitable organic solvent. Depending on the substrate, such solvents are described in the literature. According to the present invention, cholic acid and its derivatives can be isolated from the reaction mixture, for example, with ethyl acetate, optionally after acidifying the reaction mixture with, for example, HCl. A special case is the process in which the bile acid is present in aqueous solution in the form of a salt, for example, a sodium salt. In this case, precipitation of the product can be carried out by acidifying the reaction mixture. For this purpose, for example, HCl or dilute HCl can be added to the reaction mixture in sufficient amount. For example, when a pH value of 1 to 4, preferably 2 to 3, is achieved in the process, the product is present mainly in the form of a suspension. The product can then be removed from the reaction mixture by common methods, such as filtration or centrifugation. Chromatographic methods, such as column chromatography or flash chromatography, are another alternative that can be used, for example, to isolate the product. Furthermore, the product can be obtained, for example, by evaporating the reaction solvent.

[0042] Alternatively, in the process according to the invention, the product may also remain in the reaction mixture after the reaction, for example in order to carry out further reactions and optionally isolate the final product upon completion of those reactions. It is also conceivable that one or more substrates for the process according to the invention are produced in the same reaction batch by previous or parallel reactions.

[0043] example The present invention will be explained in more detail using the following examples, but the present invention is not limited thereto.

[0044] Example 1: Testing bacterial strains The following strains were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ [Deutsche Stammsammlung fur Mikroorganismen und Zellkulturen]): Saccharothrix longispora (DSM-43749), Catellatospora citrae (DSM-44097), Streptomyces hygroscopicus subsp. hygroscopicus (DSM-40578), and Asanoa ferruginea (DSM-44099). The strains were cultivated under standard conditions recommended by the DSMZ. As soon as the cultures reached visible turbidity, deoxycholate (0.5 mM) was added and the cultures were further cultivated for up to 72 h. After a centrifugation step, the culture supernatant was extracted with ethyl acetate and analyzed by HPLC and GC / MS. The HPLC chromatogram of the reaction with Streptomyces hygroscopicus showed a peak whose retention time corresponded to that of ursocholic acid. GC / MS analysis indicated that the potential ursocholic acid peak was derived from a bile acid with three hydroxyl groups. Examination of other strains did not reveal the 7-hydroxylation product of deoxycholic acid.

[0045] Example 2: Genome sequencing and annotation of P450 genes Genomic DNA of Streptomyces hygroscopicus subsp. hygroscopicus (DSM-40578) was isolated from the strain by culturing it according to the DSMZ protocol (Kieser et al. (2000), Practical Streptomyces Genetics, Norwich: John Innes Foundation). Genome sequencing was performed using an Illumina MiSeq and assembly was performed based on the known genome of Streptomyces rapamycinicus (Microsynth GmbH, Switzerland). Homology comparison allowed the identification of 42 P450 genes.

[0046] Example 3: Cloning of expression systems The following constructs containing the coding regions of putidaredoxin reductase (PtR) and putidaredoxin (Ptx) were cloned into the plasmid pJ411 (DNA2.0) using the restriction enzyme XhoI.

[0047] Synthetic DNA (Life Technologies): 5', XhoI interface, HindIII interface, approximately 50 bp spacer DNA, ribosome binding site (rbs), ORF (open reading frame) putidaredoxin reductase (PtR), approximately 50 bp spacer DNA, rbs, ORF putidaredoxin (Ptx), XhoI interface, 3'.

[0048] The results of the cloning process were confirmed by restriction enzyme digestion and DNA sequencing.

[0049] Subsequently, the ORFs encoding the P450 hydroxylases identified in Example 2 were cloned into the synthetic DNA and the plasmid, respectively, using the restriction enzymes NdeI and HindIII (Life Technologies). The results were again verified by restriction enzyme digestion and DNA sequencing. The expression vector and redox partner used in this example constitute only one method for expressing cytochrome P450 enzymes according to the present invention, and this method is chosen as an example.

[0050] Expression plasmids (see Example 2) generated using the identified P450 candidates can be used to co-express each P450 protein together with putidaredoxin reductase and putidaredoxin. The three ORFs in each expression plasmid are expressed under the control of a T7 promoter on a common mRNA, but as separate polypeptides.

[0051] Example 4: Expression of P450 / Ptx / PtR After sequencing the Streptomyces hygroscopicus subsp. hygroscopicus genome, 42 P450 sequences were examined as potential candidates for deoxycholate 7-hydroxylase. To identify the enzyme of interest, the candidate ORFs were cloned into the expression system described in Example 3 and into the pJ411 (DNA2.0) expression vector without the coding regions for putidaredoxin reductase (PtR) and putidaredoxin (Ptx). The following protocol was used for expression:

[0052] TB-P450 Expression Medium: Terrific broth (TB) medium + 50μg / ml kanamycin + 0.5 mM 5-aminolevulinic acid (from 100x parent solution) + 1 mM thiamine (from 100x parent solution) + 1 mM MgCl2 + 2.5 mM ammonium sulfate + 50 μM FeCl3 (from 100x parent solution) +0.5mM IPTG (from 1M parent solution) (Each additive was sterile filtered through a 0.2 μm filter.) P450 lysis buffer: 100mM Tris pH 7.5 20% (v / v) glycerin 1mg / ml lysozyme

[0053] Constructs for candidate P450s to be tested were transformed into the E. coli expression strain BL21(DE3). An overnight culture was inoculated from a single colony (lysogeny broth (LB) + kanamycin). The following day, a 1:100 expression culture was inoculated (150 ml of terrific broth (TB)-P450 expression medium) and initially shaken at 37°C for 3 h in a baffled flask (1 L). The temperature was then reduced to 24°C and shaken for an additional 22 h. The culture was harvested by centrifugation at 5000g for 10 min, washed once with 0.9% (w / v) NaCl, and the pellet was frozen at -80°C. The cell pellet was thawed, weighed, resuspended in an equal volume of P450 lysis buffer, incubated on ice for 1 h, and then digested using a sonicator. After centrifugation (30 min, 21000g), the supernatant was used for the test reaction.

[0054] Example 5: Testing P450 candidates for DA hydroxylation Reaction mixture: 10-80 μl of 100 mM NADH (redox cofactor) 250 μl 1 M Tris-HCl pH 7.5 17.5 μl glycerin (50%) 100 μl of 50 mM deoxycholate solution, pH 8.5 (final 10 mM) 50 μl of E. coli lysate P450 / PtR / Ptx (see Example 4) 17.5 to 87.5 μL dH2O

[0055] The reaction mixture was placed in a 1.5 ml screw-top bottle and covered with aluminum foil. Several holes were punctured in the foil. The mixture was gently shaken at 24°C for 18 hours. A 200 μl reaction batch was diluted with 600 μl acetonitrile / 5 μl H3PO4 (50%) and incubated at 55°C for 15 minutes. The sample was then centrifuged at 20817 rcf for 5 minutes and analyzed by HPLC / DAD (e.g., Agilent 1200 series, column: Merck Purospher STAR RP-18e 125 × 4 mm, 5 μm, The analysis was performed using a flow rate of 1.5 ml / min with a gradient of H2O + H3PO4 (pH = 2.6) / acetonitrile. One of the investigated candidates ("P450_c866") was able to hydroxylate deoxycholic acid to ursocholic acid. The deoxycholic acid used was transformed in this process (see table below). The identity of the product ursocholic acid was verified by GC / MS analysis and 2D NMR. [Table 1]

[0056] In this example, the redox cofactor (NADH) is oxidized by the P450 / Ptx / PtR reaction. [Sequence List Free Text]

[0057] Sequence Listing 5 <223> E. coli codon-optimized SEQ ID NO:3 Sequence Listing 6 <223> E. coli codon-optimized SEQ ID NO:4 Sequence Listing 7 <223> Xaa is methionine or no amino acid Sequence Listing 9 <223> n is A or no nucleotide Sequence Listing 9 <223> n is T or no nucleotide Sequence Listing 9 <223> n is G or no nucleotide Sequence Listing 11 <223> E. coli codon-optimized SEQ ID NO:9 Sequence Listing 11 <223> n is A or no nucleotide Sequence Listing 11 <223> n is T or no nucleotide Sequence Listing 11 <223> n is G or no nucleotide Sequence Listing 12 <223> E. coli codon-optimized SEQ ID NO: 10

Claims

1. Use of cytochrome P450 to hydroxylate deoxycholic acid at the 7-position, comprising: The cytochrome P450 enzyme comprises the amino acid sequence of SEQ ID NO: 1 or 2.

2. A method for preparing a steroid, comprising: A method comprising converting deoxycholic acid with a cytochrome P450 enzyme, wherein the cytochrome P450 enzyme comprises the amino acid sequence of SEQ ID NO: 1 or 2.

3. 3. The method according to claim 2, characterized in that the enzymatic conversion is carried out in the presence of at least one ferredoxin and / or at least one ferredoxin reductase.

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