METHOD FOR THE HYDROXYLATION OF STEROIDS

Abstract

The present invention relates to an enzme and method for the hydroxylation of a 7-deoxysteroid having the general formula (I)

##STR00001##

at position 7 to a steroid having the general formula (II)

##STR00002##

Claims

1. (canceled)

2. A method of preparing a steroid having the general formula (II): ##STR00007## wherein X.sub.1 and X.sub.2 are independently H, Cl, F, Br, I, CF.sub.3, a C.sub.1 to C.sub.6 alkyl radical, OH, a C.sub.1 to C.sub.6 alkoxy radical, CN, NO.sub.2, N(R.sub.6).sub.2, an epoxy group, CHO, or a CO.sub.2R.sub.6 radical, wherein R.sub.6 is C(O)H, C(O)CH.sub.3, C(O)CH.sub.2CH.sub.3, C(O)(CH.sub.2).sub.2CH.sub.3, C(O)CH(CH.sub.3).sub.2, C(O)(CH.sub.2).sub.3CH.sub.3, C(O)CH(CH.sub.3)CH.sub.2CH.sub.3, C(O)CH.sub.2CH.sub.2(CH.sub.3).sub.2, C(O)C(CH.sub.3).sub.3, C(O)Ph, or C(O)CH.sub.2Ph, R.sub.1 and R.sub.2 are independently H, OH, OR.sub.7, or O, wherein R.sub.7 is C(O)H, C(O)CH.sub.3, C(O)CH.sub.2CH.sub.3, C(O)(CH.sub.2).sub.2CH.sub.3, C(O)CH(CH.sub.3).sub.2, C(O)(CH.sub.2).sub.3CH.sub.3, C(O)CH(CH.sub.3)CH.sub.2C.sub.13, C(O)CH.sub.2CH-.sub.2(CH.sub.3).sub.2, C(O)C(CH.sub.3).sub.3, C(O)Ph, or C(O)CH.sub.2Ph, R.sub.3 is H, OH, OR.sub.8, a C.sub.1 to C.sub.10 alkyl radical, a C.sub.1 to C.sub.10 alkenyl radical, CHO, C(O)(CH.sub.3), C(O)(CH.sub.2OH), CH(CH.sub.3)C(O)CH.sub.3, CH(CH.sub.3)((CH.sub.2).sub.2CO.sub.2R.sub.9), or CH(CH.sub.3)((CH.sub.2).sub.2CONHR.sub.9), wherein R.sub.8 is C(O)H, C(O)CH.sub.3, C(O)CH.sub.2CH.sub.3, C(O)(CH.sub.2).sub.2CH.sub.3, C(O)CH(CH.sub.3).sub.2, C(O)(CH.sub.2).sub.3CH.sub.3, C(O)CH(CH.sub.3)CH.sub.2CH.sub.3, C(O)CH.sub.2CH.sub.2(CH.sub.3).sub.2, C(O)C(CH.sub.3).sub.3, C((O)Ph, or C(O)CH.sub.2Ph, and R.sub.9 is CH.sub.3, CH.sub.2COOH, CH.sub.2CH.sub.3, CH(CH.sub.3).sub.2, (CH.sub.2).sub.2(C.sub.3, (CH.sub.2).sub.2SO.sub.3H, C(CH.sub.3).sub.3, (CH.sub.2).sub.3CH.sub.3, CH(CH.sub.3)CH.sub.2CH.sub.3, CH.sub.2CH.sub.2(CH.sub.3).sub.2, an aryl group, or an alkylaryl group, R.sub.4 is H, OH, or OR.sub.10, wherein R.sub.10 is C(O)H, C(O)CH.sub.3, C(O)CH.sub.2CH.sub.3, C(O)(CH.sub.2).sub.2CH.sub.3, C(O)CH(CH.sub.3).sub.2, C(O)(CH.sub.2).sub.3CH.sub.3, C(O)CH(CH.sub.3)CH.sub.2CH.sub.3, C(O)CH.sub.2CH.sub.2(CH.sub.3).sub.2, C(O)C(CH.sub.3).sub.3, C(O)Ph, or C(O)CH.sub.2Ph, and R.sub.5 is H, CF.sub.3, a C.sub.1 to C.sub.6 alkyl radical, a C.sub.1 to C.sub.6 alkenyl radical, OH, O, or a C.sub.1 to C.sub.6 alkoxy radical, wherein the dashed line denotes an optional double bond, with the proviso that the B ring has no double bond if the A ring has a C.sub.4-C.sub.5 double bond, and the C ring has no double bond if X.sub.1 and X.sub.2 form an epoxy group, or wherein the steroid having the general formula (II) is selected from the group consisting of 3?,7?,12?-trihydroxy-5?-cholane-24-acid, 3?,7?,12?-trihydroxy-5?-cholane-24-acid, 3?,7?,12?-trihydroxy-5?-cholane-24-acid, 3?,7?,12?-trihydroxy-5?-cholane-24-acid, 7?,12?-dihydroxy-3-keto-5?-cholane-24-acid, 7?,12?-dihydroxy-3-keto-5?-cholane-24-acid, 3p,7?-diydroxy-12-keto-5?-cholane-24-acid, 3?,7?-dihydroxy-5?-cholane-24-acid, 7?-hydroxy-3-keto-5?-cholane-24-acid and 3?,7?-dihydroxy-5?-cholane-24-acid, the method comprising the step of converting a 7-deoxysteroid selected from the group consisting of 3?,12?-dihydroxy-5?-cholane-24-acid, 3?,12?-dihydroxy-5?-cholane-24-acid, 3?,12?-dihydroxy-5?-cholane-24-acid, 3p,12?-dihydroxy-5?-cholane-24-acid, 3?-hydroxy-12-keto-5?-cholane-24-acid, 3-keto,12p-hydroxy-5?-cholane-24-acid, 3-keto,12?-hydroxy-5?-cholane-24-acid, 3?-hydroxy-5?-cholane-24-acid, 3-keto-5?-cholane-24-acid and 3?-hydroxy-5?-cholane-24-acid or having the general formula (I) with cytochrome P450 enzyme: ##STR00008## wherein the cytochrome P450 enzyme comprises an amino acid sequence which has an identity of at least 80% to the amino acid sequence of SEQ ID No. 1 or SEQ ID No. 2.

3. The method according to claim 2, wherein: X.sub.1, X.sub.2, R.sub.4 and R.sup.5 are H, and R.sub.1 und R.sub.2 are independently H, OH, OR.sub.7, or O, wherein R.sub.7_is C(O)H, C(O)CH.sub.3, C(O)CH.sub.2CH.sub.3, C(O)(CH.sub.2).sub.2CH.sub.3, C(O)CH(CH.sub.3).sub.2, C(O)(CH.sub.2).sub.3CH.sub.3, C(C)CH(C H.sub.3)C.sub.1H.sub.2CH.sub.13 C(O)CH.sub.2CH(CH.sub.3).sub.2, C(O)C(CH.sub.3).sub.3, C(O)Ph, or C(O)CH.sub.2Ph, R.sub.3 is a C.sub.1 to C.sub.10 alkyl radical, a C.sub.1 to C.sub.10 alkenyl radical, CH(CH.sub.3)((CH.sub.2).sub.2CO.sub.2R.sub.9), or CH(CH.sub.3)((CH.sub.2).sub.2CONHR.sub.9), wherein R.sub.9 is CH.sub.3, CH.sub.2COOH, CH.sub.2CH.sub.3, CH(CH3).sub.2, (CH.sub.2).sub.2CH.sub.3, (CH.sub.2).sub.2SO.sub.3HH, C(CH.sub.3).sub.3, (CH.sub.2).sub.3CH.sub.3, CH(CH.sub.3)CH.sub.2CH.sub.3, CH.sub.2CH(CH.sub.3).sub.2, an aryl group, or an alkylaryl group.

4. The method according to claim 2, wherein the aryl group is selected from the group consisting of a phenyl radical, a phenyl radical substituted with F, Cl, Br, NO.sub.2 or CH.sub.3, and a heteroaryl.

5. The method according to claim 2, wherein the alkylaryl group is selected from the group consisting of a benzyl group, a halogenated benzyl group in which the halogen is F, C.sub.1 or Br, and a benzyl group substituted with NO.sub.2.

6. The method according to claim 2, wherein R.sub.1 is OH, R.sub.2 is O or OH, R.sub.3 is CH(CH.sub.3)((CH.sub.2).sub.2CO.sub.2R.sub.9), R.sub.4 is H, and R.sub.5 is H.

7. (canceled)

8. The method according to claim 2, wherein the enzymatic conversion takes place in the presence of at least one ferredoxin.

9. The method according to claim 2, wherein the enzymatic conversion takes place in the presence of at least one ferredoxin reductase.

10. The method according to claim 2, wherein the enzymatic conversion takes place in the presence of at least one ferredoxin and at least one ferredoxin reductase.

11. The method according to claim 2, wherein the cytochrome P450 enzyme comprises an amino acid sequence which has an identity of at least 90% to the amino acid sequence of SEQ ID No. 1 or SEQ ID No. 2.

12. The method according to claim 2, wherein the cytochrome P450 enzyme comprises an amino acid sequence which has an identity of 100% to the amino acid sequence of SEQ ID No. 1 or SEQ ID No. 2.

Description

DESCRIPTION OF THE EMBODIMENTS

[0025] Cytochrome P450 and functional variants thereof are surprisingly capable of selectively hydroxylating 7-deoxysteroids, such as, e.g., 7-deoxycholic acid, and derivatives thereof at position 7.

[0026] According to the present invention, the cytochrome P450 enzyme comprises an amino acid sequence which is at least 80%, preferably at least 90%, in particular 100%, identical to the amino acid sequence SEQ ID No. 1 or 2.

[0027] Cytochromes P450 catalyze monooxygenase reactions of a large number of endogenous as well as exogenous substrates. They are involved, among other things, in the metabolism of steroids, eicosanoids, fatty acids and bile acids as well as of exogenous substrates such as drugs, insecticides and chemical carcinogens.

[0028] Cytochromes P450 according to the present invention can be used, for example, from bacteria such as actinobacteria, in particular, for example, from the genus Streptomyces. In this case, the sequences can be isolated, for example, from genomic DNA or a cDNA library using known techniques.

[0029] The cytochromes P450 according to the present invention and, respectively, their functional variants can optionally be present in their original organism or can be isolated therefrom, or they are expressed recombinantly or produced synthetically. Recombinantly expressed polypeptides are preferably used according to the invention.

[0030] Various established microorganisms can be used for the recombinant expression of enzymes according to the present invention, such as, e.g., Escherichia coli (E. coli), Bacillus subtilis, Saccharomyces cerevisiae or Pichia pastoris. Appropriate protocols in this regard are described in detail in the relevant specialist literature or are known to a person skilled in the art.

[0031] According to the present invention, enzymes/polypeptides are preferably used as proteins recombinantly overexpressed in E. coli, with the corresponding cell lysates preferably being used either without further processing/purification or after relatively simple processing steps (e.g., centrifugation, precipitation, concentration or lyophilization). After the recombinant overexpression of the enzymes used, E. coli cells can alternatively also be used in the reaction directly without cell disintegration or, for example, after a freezing/thawing cycle. Suitable expression plasmids are known to a person skilled in the art and can often be purchased commercially.

[0032] Functional variants of cytochrome P450 can be fragments or mutational variants of cytochrome P450, wherein fragments of cytochrome P450 can also be referred to as functional fragments. Functional variants of cytochrome P450 are capable of catalyzing the same reaction as the protein from which they have been derived. Whether a variant is functional, i.e., whether it catalyzes the same reaction as the protein from which it is derived, can be determined by establishing that the variant catalyzes the same reaction. For this purpose, there are established methods in the prior art or, respectively, those that are described herein. The conversion rates of substrates by the functional variants according to the invention can deviate from the conversion rates of the cytochrome P450 from which they have been derived.

[0033] Derivatives of 7-deoxysteroids comprise compounds derived from 7-deoxysteroids and having a wide variety of modifications, with one or several modifications at positions 3, 12 and 17 of the 7-deoxysteroid being particularly preferred. Such modifications preferably comprise substitutions as defined above.

[0034] According to a preferred embodiment of the present invention, X.sub.1, X.sub.2, R.sub.4 and R.sub.5 are H and [0035] R.sub.1 und R.sub.2 are independently H, OH, OR.sub.8 or O, wherein [0036] R.sub.8 is C(O)H, C(O)CH.sub.3, C(O)CH.sub.2CH.sub.3, C(O)(CH.sub.1).sub.1CH.sub.3, C(O)CH(C.sub.1H.sub.3).sub.2, C(O)(CH.sub.2).sub.3CH.sub.3. C(O)CH(CH.sub.3)CH.sub.2CH.sub.3, C(O)CH.sub.2CH.sub.2(CH.sub.3).sub.2, C(O)C(CH.sub.3).sub.3, C(O)Ph, C(O)CH.sub.2Ph, [0037] R.sub.3 is a C.sub.1 to C.sub.10 alkyl radical, a C.sub.1 to C.sub.10 alkylen radical, CH(CH.sub.3)((CH.sub.2).sub.2CO.sub.2R.sub.9) or CH(CH.sub.3)((CH.sub.2).sub.2CONHR.sub.9), wherein [0038] R.sub.9 is CH.sub.3, CH.sub.2C.sub.0OH, C-2CH.sub.3, CH(C.sub.1-13).sub.2, (C.sub.1-12).sub.2C.sub.113, (CH.sub.2).sub.2SO.sub.3H, C(CH.sub.3).sub.3, (CH.sub.2).sub.3CH.sub.3, CH(CH.sub.3)CH.sub.2CH.sub.3, CH.sub.2CH.sub.2(CH.sub.3).sub.2, an aryl group or an alkylaryl group.

[0039] According to a further preferred embodiment of the present invention, the aryl group is selected from the group consisting of a phenyl radical, a phenyl radical substituted with F, Cl, Br, NO.sub.2 or CH.sub.3 and a heteroaryl.

[0040] 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, wherein the halogen is F, C.sub.1 or Br, and a benzyl group substituted with NO.sub.2.

[0041] According to a preferred embodiment of the present invention, R.sub.1 is OH, R.sub.2 is 0 or OH, R.sub.3 is CH(CH.sub.3)((CH.sub.2).sub.2CO.sub.2R.sub.5), R.sub.4 is H, and R.sub.5 is H.

[0042] According to another preferred embodiment of the present invention, the 7-deoxysteroid having the general formula (II) is selected from the group consisting of 3?,12?-dihydroxy-5?-cholane-24-acid, 3?,12?-dihydroxy-5?-cholane-24-acid, 3?,12?-dihydroxy-5?-cholane-24-acid, 3?,12?-dihydroxy-5?-cholane-24-acid, 3?-hydroxy-12-keto-5?-cholane-24-acid, 3-keto,12?-hydroxy-5?-cholane-24-acid, 3-keto,12?-hydroxy-5?-cholane-24-acid, 3?-hydroxy-5?-cholane-24-acid, 3-keto-5?-cholane-24-acid, 30-hydroxy-5?-cholane-24-acid and esters of the respective acid.

[0043] The cytochrome P450 hydroxylase used, according to the invention, for the hydroxylation of 7-deoxysteroids and derivatives thereof having the general formula (I) to a steroid or a derivative thereof having the general formula (II) 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%, in particular 100%, identical to the amino acid sequence SEQ ID No. 1 or 2.

TABLE-US-00001 SEQIDNo.1: MLTTAETTSIAYPFNTAEGLALSERYEEARNRTGLLRVRMPYGEPAWLV TRYADARLVLGDRRFSRAEALHHDEPRQSEGRPDSGILTMDPPDHTRLR TLVAKAFTVHQVEKLRPWVRQLTHDLLDDLEAAGPPADLVDRYALPIPV GVICAMLGVPQEDRPKFRVWSDAALSTSSLSAEQFARNTDELRAYMAGL IEDHRRTPRDDIMTSLIEARDAGDRLSELELVDLCVGILVAGHETTATQ IPNFVLTLLEHPDQLRRLREDPALIQGAVEELLRFVPLGVGAAQARYAT EDIEVGGTLVRSGEPVLVAVGSANRDALRFDEPGVLNVARPTTQHLGFG HGVHHCLGAPLARLELQEALGALITRFPGLRLAGDIEWKDRMLVRGPRV MPIGW SEQIDNo.2: MPYGEPAWLVTRYADARLVLGDRRFSRAEALHHDEPRQSEGRRDSGILT MDPPDHTRLRTLVAKAFTVHQVEKLRPWVRQLTHDLLDDLEAAGPPADL VDRYALPIPVGVICAMLGVPQEDRPKFRVWSDAALSTSSLSAEQFARNT DELRAYMAGLIEDHRRTPRDDIMTSLIEARDAGDRLSELELVDLCVGIL VAGHETTATQIPNFVLTLLEHPDQLRRLREDPALIQGAVEELLRFVPLG VGAAQARYATEDIEVGGTLVRSGEPVLVAVGSANRDALRFDEPGVLNVA RPTTQHLGFGHGVHHCLGAPLARLELQEALGALITRFPGLRLAGDIEWK DRMLVRGPRVMPIGW

[0044] Amino acid sequences SEQ ID Nos. 1 and 2 are preferable encoded by nucleic acid sequences SEQ ID Nos. 3 and 4, with nucleic acid sequences SEQ ID Nos. 5 and 6 being optimized for expression in E. coli.

TABLE-US-00002 SEQIDNo.3: ATGTTGACCACAGCCGAGACGACATCCATCGCCTATCCCTTCAACACCGC CGAAGGGCTGGCGCTCAGCGAGCGTTACGAAGAGGCCAGGAACCGCACCG GACTGCTCCGGGTGCGGATGCCCTACGGTGAGCCCGCCTGGCTGGTCACG CGGTACGCCGACGCCCGGCTGGTGCTCGGCGACCGGCGCTTCAGCCGTGC GGAGGCGCTCCACCACGACGAGCCGCGGCAGTCCGAAGGCCGGCGCGACA GCGGCATCCTGACCATGGACCCGCCCGACCACACCCGGCTGCGCACCCTC GTCGCCAAGGCGTTCACCGTCCACCAGGTGGAGAAACTCCGCCCCTGGGT ACGCCAGTTGACCCATGACCTGCTCGACGACCTCGAGGCCGCCGGGCCGC CCGCCGATCTGGTGGACCGCTACGCCCTGCCCATTCCGGTCGGCGTCATC TGCGCCATGCTCGGCGTCCCGCAGGAGGACCGGCCCAAGTTCCGGGTCTG GAGCGACGCCGCGCTGTCCACCAGCTCGCTGAGCGCCGAGCAGTTCGCCC GTAACACCGACGAGCTGCGCGCCTACATGGCCGGGCTGATCGAGGACCAC CGCAGGACCCCGCGGGACGACATCATGACCTCGCTGATCGAGGCGCGGGA CGCGGGCGACCGGCTGTCCGAGCTGGAACTCGTCGATCTGTGCGTGGGCA TCCTGGTGGCCGGGCACGAGACCACCGCCACCCAGATCCCCAACTTCGTG CTGACGCTGCTGGAGCACCCGGACCAGCTGCGCCGGCTGCGCGAGGACCC CGCCCTGATCCAGGGCGCCGTCGAGGAGCTGCTGCGCTTCGTCCCGCTGG GCGTGGGCGCCGCCCAGGCCCGTTACGCCACCGAGGACATCGAGGTGGGC GGCACGCTGGTGCGCAGCGGGGAGCCGGTGCTGGTCGCCGTCGGCTCGGC CAACCGCGACGCGCTGCGCTTCGACGAACCGGGCGTGCTCAACGTCGCCC GCCCCACCACCCAGCACCTCGGCTTCGGCCACGGTGTGCACCACTGCCTG GGCGCGCCCCTGGCCCGTCTGGAGCTCCAGGAGGCGCTCGGCGCGCTGAT CACGCGCTTCCCGGGCCTGCGGCTGGCCGGGGACATCGAGTGGAAGGACC GCATGCTGGTCCGCGGGCCCCGTGTCATGCCCATCGGGTGGTGA SEQIDNo.4: ATGCCCTACGGTGAGCCCGCCTGGCTGGTCACGCGGTACGCCGACGCCCG GCTGGTGCTCGGCGACCGGCGCTTCAGCCGTGCGGAGGCGCTCCACCACG ACGAGCCGCGGCAGTCCGAAGGCCGGCGCGACAGCGGCATCCTGACCATG GACCCGCCCGACCACACCCGGCTGCGCACCCTCGTCGCCAAGGCGTTCAC CGTCCACCAGGTGGAGAAACTCCGCCCCTGGGTACGCCAGTTGACCCATG ACCTGCTCGACGACCTCGAGGCCGCCGGGCCGCCCGCCGATCTGGTGGAC CGCTACGCCCTGCCCATTCCGGTCGGCGTCATCTGCGCCATGCTCGGCGT CCCGCAGGAGGACCGGCCCAAGTTCCGGGTCTGGAGCGACGCCGCGCTGT CCACCAGCTCGCTGAGCGCCGAGCAGTTCGCCCGTAACACCGACGAGCTG CGCGCCTACATGGCCGGGCTGATCGAGGACCACCGCAGGACCCCGCGGGA CGACATCATGACCTCGCTGATCGAGGCGCGGGACGCGGGCGACCGGCTGT CCGAGCTGGAACTCGTCGATCTGTGCGTGGGCATCCTGGTGGCCGGGCAC GAGACCACCGCCACCCAGATCCCCAACTTCGTGCTGACGCTGCTGGAGCA CCCGGACCAGCTGCGCCGGCTGCGCGAGGACCCCGCCCTGATCCAGGGCG CCGTCGAGGAGCTGCTGCGCTTCGTCCCGCTGGGCGTGGGCGCCGCCCAG GCCCGTTACGCCACCGAGGACATCGAGGTGGGCGGCACGCTGGTGCGCAG CGGGGAGCCGGTGCTGGTCGCCGTCGGCTCGGCCAACCGCGACGCGCTGC GCTTCGACGAACCGGGCGTGCTCAACGTCGCCCGCCCCACCACCCAGCAC CTCGGCTTCGGCCACGGTGTGCACCACTGCCTGGGCGCGCCCCTGGCCCG TCTGGAGCTCCAGGAGGCGCTCGGCGCGCTGATCACGCGCTTCCCGGGCC TGCGGCTGGCCGGGGACATCGAGTGGAAGGACCGCATGCTGGTCCGCGGG CCCCGTGTCATGCCCATCGGGTGGTGA SEQIDNo.5: ATGCTGACCACCGCAGAAACCACCAGTATTGCATATCCGTTTAATACCGC AGAAGGTCTGGCACTGAGCGAACGTTATGAAGAAGCACGTAATCGTACCG GTCTGCTGCGTGTTCGTATGCCGTATGGTGAACCGGCATGGCTGGTTACC CGTTATGCAGATGCCCGTCTGGTTCTGGGTGATCGTCGTTTTAGCCGTGC CGAAGCACTGCATCACGATGAACCGCGTCAGAGCGAAGGTCGTCGTGATA GCGGTATTCTGACCATGGATCCGCCTGATCATACCCGTCTGCGTACCCTG GTTGCAAAAGCATTTACCGTTCATCAGGTTGAAAAACTGCGTCCGTGGGT TCGCCAGCTGACCCATGATCTGCTGGATGATCTGGAAGCAGCAGGTCCGC CTGCAGATCTGGTTGATCGTTATGCACTGCCGATTCCGGTTGGTGTTATT TGTGCAATGCTGGGTGTTCCGCAAGAAGATCGTCCTAAATTTCGTGTTTG GAGTGATGCAGCACTGAGCACCAGCAGCCTGAGCGCAGAACAGTTTGCAC GTAATACCGATGAACTGCGTGCATATATGGCAGGTCTGATTGAAGATCAT CGTCGTACACCGCGTGATGATATTATGACCAGCCTGATCGAAGCACGTGA TGCCGGTGATCGCCTGAGTGAACTGGAACTGGTGGATCTGTGTGTTGGTA TTCTGGTTGCAGGTCATGAAACCACCGCAACCCAGATTCCGAATTTTGTT CTGACCCTGCTGGAACATCCGGATCAGCTGCGTCGTCTGCGTGAAGATCC GGCACTGATTCAGGGTGCAGTTGAAGAACTGCTGCGTTTTGTTCCGCTGG GTGTGGGTGCAGCACAGGCACGTTATGCAACCGAAGATATTGAAGTTGGT GGCACCCTGGTTCGTAGTGGCGAACCGGTGCTGGTTGCCGTTGGTAGCGC AAACCGTGATGCACTGCGCTTTGATGAACCGGGTGTTCTGAATGTTGCAC GTCCGACCACACAGCATCTGGGTTTTGGTCATGGTGTTCATCATTGTCTG GGTGCACCGCTGGCACGTCTGGAACTGCAAGAAGCACTGGGAGCACTGAT TACCCGTTTTCCGGGTCTGCGTCTGGCAGGCGATATTGAATGGAAAGATC GTATGCTGGTTCGTGGTCCGCGTGTTATGCCGATTGGTTGGTAA SEQIDNo.6: ATGGTGAACCGGCATGGCTGGTTACCCGTTATGCAGATGCCCGTCTGGTT CTGGGTGATCGTCGTTTTAGCCGTGCCGAAGCACTGCATCACGATGAACC GCGTCAGAGCGAAGGTCGTCGTGATAGCGGTATTCTGACCATGGATCCGC CTGATCATACCCGTCTGCGTACCCTGGTTGCAAAAGCATTTACCGTTCAT CAGGTTGAAAAACTGCGTCCGTGGGTTCGCCAGCTGACCCATGATCTGCT GGATGATCTGGAAGCAGCAGGTCCGCCTGCAGATCTGGTTGATCGTTATG CACTGCCGATTCCGGTTGGTGTTATTTGTGCAATGCTGGGTGTTCCGCAA GAAGATCGTCCTAAATTTCGTGTTTGGAGTGATGCAGCACTGAGCACCAG CAGCCTGAGCGCAGAACAGTTTGCACGTAATACCGATGAACTGCGTGCAT ATATGGCAGGTCTGATTGAAGATCATCGTCGTACACCGCGTGATGATATT ATGACCAGCCTGATCGAAGCACGTGATGCCGGTGATCGCCTGAGTGAACT GGAACTGGTGGATCTGTGTGTTGGTATTCTGGTTGCAGGTCATGAAACCA CCGCAACCCAGATTCCGAATTTTGTTCTGACCCTGCTGGAACATCCGGAT CAGCTGCGTCGTCTGCGTGAAGATCCGGCACTGATTCAGGGTGCAGTTGA AGAACTGCTGCGTTTTGTTCCGCTGGGTGTGGGTGCAGCACAGGCACGTT ATGCAACCGAAGATATTGAAGTTGGTGGCACCCTGGTTCGTAGTGGCGAA CCGGTGCTGGTTGCCGTTGGTAGCGCAAACCGTGATGCACTGCGCTTTGA TGAACCGGGTGTTCTGAATGTTGCACGTCCGACCACACAGCATCTGGGTT TTGGTCATGGTGTTCATCATTGTCTGGGTGCACCGCTGGCACGTCTGGAA CTGCAAGAAGCACTGGGAGCACTGATTACCCGTTTTCCGGGTCTGCGTCT GGCAGGCGATATTGAATGGAAAGATCGTATGCTGGTTCGTGGTCCGCGTG TTATGCCGATTGGTTGGTAA

[0045] Identical as used herein means that two or more amino acid sequences, when superimposed on one another, may have a certain identity (matching amino acid residues at identical positions) to one another. Identity is defined in this invention as the percentage of amino acids of eligible amino acid sequences that are identical to the amino acids of the starting sequence, namely after the alignment of the two sequences and the introduction of gaps, if necessary, in order to achieve the maximum percentual sequence identity as 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. Herein, the algorithm blastp (protein-protein-BLAST) is used with the following parameters: expect threshold: 0.05; word size: 6; matrix: BLOSUM62; gap costs: Existence 11, Extension 1; conditional compositional score matrix adjustment; no filter and no mask. A percentage (%) value for the amino acid sequence identity is determined by the number of matching identical nucleotides divided by the sequence length for which the identity in percent is recorded.

[0046] A further aspect of the present invention relates to a method of preparing a steroid or a derivative thereof having the general formula (II)

##STR00005##

wherein [0047] X.sub.1 and X.sub.2 are independently H, Cl, F, Br, I, CF.sub.3, a C.sub.1 to C.sub.6 alkyl radical, OH, a C.sub.1 to C.sub.6 alkoxy radical, CN, NO.sub.2, N(R.sub.6).sub.2, an epoxy group, CHO or a CO.sub.2R.sub.6 radical, wherein [0048] R.sub.6 is C(O)H, C(O)CH.sub.3, C(O)CH.sub.2CH.sub.3, C(O)(CH.sub.2).sub.2CH.sub.3, C(O)CH(CH.sub.3).sub.2,C(O)(CH.sub.2).sub.3CH.sub.3, C(O)CH(CH.sub.3)CH.sub.2CH.sub.3, C(O)CH.sub.2CH.sub.2(CH.sub.3).sub.2, C(O)C(CH.sub.3).sub.3, C(O)Ph, C(O)CH-.sub.2Ph, R.sub.1 and R.sub.2 are independently H, OH, OR.sub.7 or O, wherein [0049] R.sub.7 is C(O)H, C(O)CH.sub.3, C(O)CH.sub.2CH.sub.3, C(O)(CH.sub.2).sub.2CH.sub.3, C(O)CH(CH.sub.3).sub.2, C(O)(CH.sub.2).sub.3CH.sub.3, (O)CH(CH.sub.3,CO)CH.sub.2CH.sub.3, C(O)CH(CH.sub.2(CH.sub.3).sub.2, C(O)C(CH.sub.3), C(O)Ph, C(O)CH.sub.2Ph, [0050] R.sub.3 is H, OH, OR.sub.8, a C.sub.1 to C.sub.10 alkyl radical, a C.sub.1 to C.sub.10 alkenyl radical, CHO, C(O)(CH.sub.3), C(O)(CH.sub.2OH)), CH(CH.sub.3)C(O)C.sub.13, CH(CH.sub.3)((CH.sub.2).sub.2CO.sub.2R.sub.9) or CH(CH.sub.3)((CH.sub.2).sub.2CONHR.sub.9), wherein [0051] R.sub.8 is C(O)H, C(O)CH.sub.3, C(O)CH.sub.2CH.sub.3, C(O)(CH.sub.2).sub.2CH.sub.3, C(O)CH(CH.sub.3).sub.2, C(O)(CH.sub.2).sub.3CH.sub.3, C(O)CH(CH.sub.3)CH.sub.2CH.sub.3, C(O)CH.sub.2CH.sub.2(CH.sub.3).sub.2, C(O)C(CH.sub.3).sub.3, C(O)Ph or C(O)CH.sub.2Ph, and [0052] R.sub.9 is CH.sub.3, CH.sub.2COOH, CH.sub.2CH.sub.3, CH(CH.sub.3).sub.2, (CH.sub.2).sub.2CH.sub.3, (CH.sub.2).sub.2S03H, C(CH.sub.3).sub.3, (CH.sub.2).sub.3CH.sub.3, CH(CH.sub.3)CH.sub.2CH.sub.3, CH.sub.2CH.sub.2(CH.sub.3).sub.2, an aryl group or an alkylaryl group, R.sub.4 is H, OH, or OR.sub.10, wherein [0053] R.sub.10 is C(O)H, C(O)CH.sub.3, C(O)CH.sub.2CH.sub.3, C(O)(CH.sub.2).sub.2CH.sub.3, C(O)CH(CH.sub.3).sub.2, C(O)(CH.sub.2).sub.3C.sub.1-3, C(O)CH(CH.sub.3)CH.sub.2CH.sub.3, C(O)CH.sub.2CH.sub.2(CH.sub.3).sub.2, C(O)C(CH.sub.3).sub.3, C(O)Ph or C(O)CH.sub.2Ph, and [0054] R.sub.5 is H, CF.sub.3, a C.sub.1 to C.sub.6 alkyl radical, a C.sub.1 to C.sub.6 alkenyl radical, OH, O, or a C.sub.1 to C.sub.6 alkoxy radical, wherein the dashed line denotes an optional double bond, with the proviso that the B ring has no double bond if the A ring has a C.sub.4-C.sub.5 double bond, and the C ring has no double bond if X.sub.1 and X.sub.2 form an epoxy group, [0055] comprising the step of converting a 7-deoxysteroid or a derivative thereof having the general formula (I)

##STR00006##

with cytochrome P450 or a functional variant thereof, characterized in that the cytochrome P450 enzyme comprises an amino acid sequence which is at least 80%, preferably at least 90%, in particular 100%, identical to the amino acid sequence SEQ ID No. 1 or 2.

[0056] Using the method according to the invention, 7-deoxysteroids or, respectively, derivatives thereof having the general formula (I) can be converted with cytochrome P450 according to the invention or a functional variant thereof to steroids or, respectively, derivatives thereof having the general formula (II).

[0057] In order to support the redox reaction of cytochrome P450 according to the invention or, respectively, its functional variants thereof, it is advantageous to carry out the method according to the invention in the presence of a reducing agent. NAD(P)H, flavins or ferredoxins can be used as reducing agents. For example, if the redox cofactors NAD(P)+ and/or NAD(P)H are used, it is advantageous to use them at a concentration of 0.001 mM and 10 mM, more preferably between 0.05 mM and 1 mM, in a reaction mixture.

[0058] The method according to the invention is preferably carried out in the presence of redox partners for cytochrome P450. Redox partners are understood to be proteins of the ferredoxin and ferredoxin reductase classes, which are advantageous for the function of cytochrome P450 according to the present invention. A possible pair of redox partners preferably comprises putidaredoxin and putidaredoxin reductase from Pseudomonas putida. Moreover, a person skilled in the art is able to identify further ferredoxin proteins and ferredoxin reductases which are potential redox partners for the cytochrome P450 according to the invention. Suitability as a redox partner can be verified in a functional assay, as described, for example, in Examples 3 to 5. The putidaredoxin used in these examples and/or the putidaredoxin reductase used therein can be replaced by possible alternative proteins or enzymes, respectively. If sufficient formation of the desired product (e.g., ursocholic acid) is observed, the tested redox partners can be regarded as functional alternatives to putidaredoxin and/or putidaredoxin reductase.

[0059] According to a particularly preferred embodiment of the present 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%, in particular 100%, identical to the amino acid sequence SEQ ID No. 7, wherein X is a methionine residue or is not an amino acid.

TABLE-US-00003 SEQIDNo.7: XSKVVYVSHDGTRRELDVADGVSLMQAAVSNGIYDIVGDCGGSASCAT CHVYVNEAFTDKVPAANEREIGMLECVTAELKPNSRLCCQIIMTPELD GIVVDVPDRQW

[0060] According to a further preferred embodiment of the present 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%, in particular 100%, identical to the amino acid sequence SEQ ID No. 8.

TABLE-US-00004 SEQIDNo.8: MNANDNVVIVGTGLAGVEVAFGLRASGWEGNIRLVGDATVIPHHLPPL SKAYLAGKATAESLYLRTPDAYAAQNIQLLGGTQVTAINRDRQQVILS DGRALDYDRLVLATGGRPRPLPVASGAVGKANNFRYLRTLEDAECIRR QLIADNRLVVIGGGYIGLEVAATAIKANMHVTLLDTAARVLERVTAPP VSAFYEHLHREAGVDIRTGTQVCGFEMSTDQQKVTAVLCEDGTRLPAD LVIAGIGLIPNCELASAAGLQVDNGIVINEHMQTSDPLIMAVGDCARF HSQLYDRWVRIESVPNALEQARKIAAILCGKVPRDEAAPWFWSDQYEI GLKMVGLSEGYDRIIVRGSLAQPDFSVFYLQGDRVLAVDTVNRPVEFN QSKQIITDRLPVEPNLLGDESVPLKEIIAAAKAELSSA

[0061] Amino acid sequences SEQ ID Nos. 7 and 8 are preferably encoded by nucleic acid sequences SEQ ID Nos. 9 and 10, respectively, with nucleic acid sequences SEQ ID Nos. 11 and 12 being optimized for expression in E. coli.

TABLE-US-00005 SEQIDNo.9: (ATG).sub.0or1 TCTAAAGTAGTGTATGTGTCACATGATGGAACGCGTCGCGAACTGGATG TGGCGGATGGCGTCAGCCTGATGCAGGCTGCAGTCTCCAATGGTATCTA CGATATTGTCGGTGATTGTGGCGGCAGCGCCAGCTGTGCCACCTGCCAT GTCTATGTGAACGAAGCGTTCACGGACAAGGTGCCCGCCGCCAACGAGC GGGAAATCGGCATGCTGGAGTGCGTCACGGCCGAACTGAAGCCGAACAG CAGGCTCTGCTGCCAGATCATCATGACGCCCGAGCTGGATGGCATCGTG GTCGATGTTCCCGATAGGCAATGGTAA SEQIDNo.10: ATGAACGCAAACGACAACGTGGTCATCGTCGGTACCGGACTGGCTGGCG TTGAGGTCGCCTTCGGCCTGCGCGCCAGCGGCTGGGAAGGCAATATCCG GTTGGTGGGGGATGCGACGGTAATTCCCCATCACCTACCACCGCTATCC AAAGCTTACTTGGCCGGCAAAGCCACAGCGGAAAGCCTGTACCTGAGAA CCCCAGATGCCTATGCAGCGCAGAACATCCAACTACTCGGAGGCACACA GGTAACGGCTATCAACCGCGACCGACAGCAAGTAATCCTATCGGATGGC CGGGCACTGGATTACGACCGGCTGGTATTGGCTACCGGAGGGCGTCCAA GACCCCTACCGGTGGCCAGTGGCGCAGTTGGAAAGGCGAACAACTTTCG ATACCTGCGCACACTCGAGGACGCCGAGTGCATTCGCCGGCAGCTGATT GCGGATAACCGTCTGGTGGTGATTGGTGGCGGCTACATTGGCCTTGAAG TGGCTGCCACCGCCATCAAGGCGAACATGCACGTCACCCTGCTTGATAC GGCAGCCCGGGTTCTGGAGCGGGTTACCGCCCCGCCGGTATCGGCCTTT TACGAGCACCTACACCGCGAAGCCGGCGTTGACATACGAACCGGCACGC AGGTGTGCGGGTTCGAGATGTCGACCGACCAACAGAAGGTTACTGCCGT CCTCTGCGAGGACGGCACAAGGCTGCCAGCGGATCTGGTAATCGCCGGG ATTGGCCTGATACCAAACTGCGAGTTGGCCAGTGCGGCCGGCCTGCAGG TTGATAACGGCATCGTGATCAACGAACACATGCAGACCTCTGATCCCTT GATCATGGCCGTCGGCGACTGTGCCCGATTTCACAGTCAGCTCTATGAC CGCTGGGTGCGTATCGAATCGGTGCCCAATGCCTTGGAGCAGGCACGAA AGATCGCCGCCATCCTCTGTGGCAAGGTGCCACGCGATGAGGCGGCGCC CTGGTTCTGGTCCGATCAGTATGAGATCGGATTGAAGATGGTCGGACTG TCCGAAGGGTACGACCGGATCATTGTCCGCGGCTCTTTGGCGCAACCCG ACTTCAGCGTTTTCTACCTGCAGGGAGACCGGGTATTGGCGGTCGATAC AGTGAACCGTCCAGTGGAGTTCAACCAGTCAAAACAAATAATCACGGAT CGTTTGCCGGTTGAACCAAACCTACTCGGTGACGAAAGCGTGCCGTTAA AGGAAATCATCGCCGCCGCCAAAGCTGAACTGAGTAGTGCCTGA SEQIDNo.11: (ATG).sub.0or1 ATGAGCAAAGTGGTCTATGTGTCGCATGATGGAACACGCCGTGAGTTAG ACGTCGCTGATGGTGTATCCCTGATGCAAGCAGCGGTTAGCAATGGCAT TTACGACATCGTTGGCGATTGTGGTGGTAGTGCGTCATGTGCAACGTGT CACGTGTATGTTAACGAAGCGTTTACCGATAAGGTGCCTGCTGCCAATG AACGCGAGATTGGCATGCTGGAATGCGTAACTGCCGAACTCAAACCGAA CTCTCGCCTGTGCTGCCAGATCATCATGACCCCGGAATTGGACGGGATT GTCGTTGATGTGCCAGATCGTCAGTGGTAA SEQIDNo.12: ATGAACGCCAATGATAATGTTGTTATTGTTGGCACCGGTCTGGCAGGCG TTGAAGTTGCATTTGGTCTGCGTGCAAGCGGTTGGGAAGGTAATATTCG TCTGGTTGGTGATGCAACCGTTATTCCGCATCATCTGCCTCCGCTGAGC AAAGCATATCTGGCAGGTAAAGCAACCGCAGAAAGCCTGTATCTGCGTA CACCGGATGCCTATGCAGCACAGAATATTCAGCTGCTGGGTGGTACACA GGTTACCGCAATTAATCGTGATCGTCAGCAGGTTATTCTGAGTGATGGT CGTGCACTGGATTATGATCGTCTGGTGCTGGCAACCGGTGGTCGTCCGC GTCCGCTGCCGGTTGCAAGTGGTGCAGTTGGTAAAGCCAATAACTTTCG TTATCTGCGCACCCTGGAAGATGCAGAATGTATTCGTCGTCAGCTGATT GCAGATAATCGCCTGGTTGTGATTGGTGGTGGTTATATTGGTCTGGAAG TTGCAGCAACCGCCATTAAAGCAAATATGCATGTTACCCTGCTGGATAC CGCAGCACGTGTTCTGGAACGTGTTACCGCACCGCCTGTTAGCGCCTTT TATGAACATCTGCATCGTGAAGCCGGTGTTCATATTCGTACCGGCACCC AGGTTTGTGGTTTTGAAATGAGCACCGATCAGCAGAAAGTTACCGCAGT TCTGTGTGAAGATGGCACCCGTCTGCCTGCAGATCTGGTTATTGCAGGT ATTGGCCTGATTCCGAATTGTGAACTGGCAAGCGCAGCAGGTCTGGCAG TTGGTGATTGTGCACGTTTTCATAGCCAGCTGTATGATCGTTGGGTTCG TATTGAAAGCGTTCCGAATGCACTGGAACAGGCACGTAAAATTGCAGCA ATTCTGTGTGGTAAAGTTCCGCGTGATGAAGCAGCACCGTGGTTTTGGA GCGATCAGTATGAAATTGGTCTGAAAATGGTTGGTCTGAGCGAAGGTTA TGATCGCATTATTGTTCGTGGTAGCCTGGCACAGCCGGATTTTTCAGTT TTTTATCTGCAGGGTGATCGTGTGCTGGCAGTTGATACCGTTAATCGTC CGGTTGAATTTAACCAGAGCAAACAAATTATCACCGATCGTCTGCCGGT GGAACCGAATCTGCTGGGAGATGAAAGCGTGCCGCTGAAAGAAATTATT GCAGCAGCAAAAGCAGAACTGAGCAGCGCATAA

[0062] The expression of the cytochrome P450 according to the invention and any ferredoxins and ferredoxin reductases in bacteria, in particular in E. coli, is particularly advantageous when nucleic acids with the nucleic acid sequences SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 11 and/or SEQ ID No. 12 are used. Further aspects of the present invention therefore relate to a nucleic acid (DNA and/or RNA) with 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 and vectors and/or cells, in particular E. coli cells, comprising at least one of those sequences.

[0063] It has been shown that it is particularly advantageous if the above-mentioned ferredoxins and ferredoxin reductases are expressed (co-expressed) together with cytochrome P450 in a production strain (e.g., an E. coli strain). Through the co-expression of the three proteins or, respectively, enzymes, ideally under the same promoter, an ideal balance between the enzymes can be established, which has a particularly advantageous effect on the enzymatic conversion of a substrate.

[0064] According to a preferred embodiment of the present invention, the method according to the invention is performed at a temperature of from 10? to 40? C., preferably from 15? to 38? C., more preferably from 20? C. to 30? C., more preferably from 22? C. to 26? C. It has been shown according to the invention that the enzyme activity ofcytochrome P450 for the reaction according to the invention is particularly high in this area.

[0065] According to a further preferred embodiment of the present invention, the method according to the invention is performed at a pH offrom 6.5 to 8.5, preferably from 7 to 8, more preferably from 7.2 to 7.8. At this pH value, the enzyme activity of cytochrome P450 is highest so as to allow an appropriate conversion of the substrate.

[0066] The hydroxylation of deoxysteroids or, respectively, deoxysteroid derivatives can be carried out regioselectively at position 7 of the steroid backbone. In this way, in particular, a 7beta-hydroxyl group can be introduced stereoselectively so that, for example, ursocholic acid and/or ursocholic acid derivatives can be produced.

[0067] In the method according to the present invention, the isolation of the product can be effected in different ways. For example, the product can be extracted from the reaction mixture by a suitable organic solvent. Depending on the substrate, such solvents are described in the literature. According to the present invention, cholic acids and their derivatives can be isolated from reaction mixtures, for example, with ethyl acetate, optionally after acidification of the reaction mixture, e.g., with HCl. A method in which bile acids are present in the form of a salt, e.g., a sodium salt, in an aqueous solution constitutes a special case. In this case, a precipitation of the product can be effected by acidifying the reaction mixture. For this purpose, for example, HCl or dilute HCl can be added to the reaction mixture in a sufficient amount. If a pH value of, for example, 1 to 4, preferably 2 to 3, is achieved in the process, the product predominantly exists in the form of a suspension. The product can then be removed from the reaction mixture by common methods such as, e.g., filtration or centrifugation. Chromatographic methods, such as, e.g., column chromatography or flash chromatography, are another alternative that can be used for product isolation, for example. Furthermore, it is possible, for example, to obtain product by evaporating the reaction solvent.

[0068] Alternatively, in a method according to the present invention, the product(s) may also remain in the reaction mixture after the reaction, e.g., in order to carry out even more reactions and optionally isolate an end product upon completion of those reactions. It is also conceivable that the substrate(s) for the method according to the present invention is/are produced in the same reaction batch by previous reactions or reactions taking place in parallel.

EXAMPLES

[0069] The present invention is explained in further detail using the following examples, without, however, being restricted thereto.

Example 1: Test of Bacterial Strains

[0070] The following bacterial strains were obtained from the German Strain Collection of Microorganisms and Cell Cultures (DSMZ [Deutsche Stammsammlung f?r 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 as recommended by DSMZ. As soon as the growth of the cultures had led to visible turbidity, deoxycholic acid (0.5 mM) was added, and it was cultured further for up to 72 h. After a centrifugation step, supernatants of the cultures were extracted with ethyl acetate and analyzed by HPLC and GC/MS. In the HPLC chromatogram of the reaction with Streptomyces hygroscopicus, a peak was noted the retention time of which corresponds to that of ursocholic acid. The GC/MS analysis indicated that the potential ursocholic acid peak originates from a bile acid with 3 hydroxyl groups. The examination of the other strains gave no indication of 7-hydroxylated products of deoxycholic acid.

Example 2: Genome Sequencing and Annotation of the P450 Genes

[0071] Upon cultivation of Streptomyces hygroscopicus subsp. hygroscopicus (DSM-40578) according to the DSMZ regulation, the genomic DNA of the strain was isolated (Kieser et al. (2000), Practical Streptomyces genetics (Norwich: John Innes Foundation)). The genome was sequenced using Illumina MiSeq, and an assembly based on the known genome of Streptomyces rapamycinicus was conducted (Microsynth GmbH, Switzerland). 42 P450 genes could be identified by homology comparisons.

Example 3: Cloning of Expression System

[0072] Using the restriction enzyme XhoI, the following construct comprising coding regions for putidaredoxin reductase (PtR) and putidaredoxin (Ptx) was cloned into plasmid pJ411 (DNA 2.0).

[0073] Synthetic DNA (Life Technologies): 5, XhoI interface, HindIII interface, approx. 50 bp spacer DNA, ribosome binding site (rbs), ORF (open reading frame) putidaredoxin reductase (PtR), approx. 50 bp spacer DNA, rbs, ORF putidaredoxin (Ptx), XhoI interface, 3.

[0074] The result of the cloning step was checked by means of restriction enzyme digestion and DNA sequencing.

[0075] Subsequently, using the restriction enzymes NdeI and HindIII, one ORF each coding for the P450 hydroxylases identified in Example 2 was cloned into the above-mentioned synthetic DNA or plasmid, respectively (Life Technologies). The result was again verified by means of restriction enzyme digestion and DNA sequencing. The expression vector used in this example and the redox partners used constitute only one way of expressing the cytochrome P450 enzymes according to the invention, which way has been chosen as an example.

[0076] The expression plasmids produced with the identified P450 candidates (see example 2) can be used for jointly expressing the respective P450 proteins together with putidaredoxin reductase and putidaredoxin. The 3 ORFs of the respective expression plasmids are expressed under the control of a T7 promoter on a common mRNA, but as separate polypeptides.

Example 4: Expression of P450/Ptx/PtR

[0077] After the genome sequencing of Streptomyces hygroscopicus subsp. hygroscopicus, there were 42 P450 sequences that came into consideration as candidates for a possible deoxycholic acid-7-hydroxylase. To identify the enzyme looked for, ORFs of the candidates were cloned into the expression system described in example 3 and into a pJ411 (DNA 2.0) expression vector without coding regions for putidaredoxin reductase (PtR) and putidaredoxin (Ptx). The following protocol was used for the expression.

[0078] TB-P450 expression medium: [0079] Terrific broth (TB) medium [0080] +50 ?g/ml kanamycin [0081] +0.5 mM 5-aminolevulinic acid (from 100?parent solution) [0082] +1 mM thiamine (from 100?parent solution) [0083] +1 mM MgCl.sub.2+2.5 mM ammonium sulfate+50 ?M FeCl.sub.3 (from 100?parent solution) [0084] +0.5 mM IPTG (from 1 M parent solution) [0085] (the additives were each 0.2 ?m sterile filtered)

[0086] P450 lysis buffer: [0087] 100 mM Tris pH 7.5 [0088] 20% (v/v) glycerin [0089] 1 mg/ml lysozyme

[0090] The constructs of the P450 candidates, which were to be tested, were transformed into the E. coli expression strain BL21 (DE3). Overnight cultures were inoculated from single colonies (LB (lysogeny broth)+kanamycin). The next day, 1:100 expression cultures were inoculated therewith (150 ml TB (terrific broth) -P450 expression medium) and were initially shaken at 370? C. in baffled flasks (1 L) for 3 h. Subsequently, the temperature was lowered to 240? C., and it was shaken for another 22 h. The cultures were harvested by centrifugation at 5000 g for 10 min, washed 1x with 0.9% (w/v) NaCl, and pellets were frozen at ?80? C. The cell pellets were thawed, weighed and resuspended with an equivalent amount of P450 lysis buffer, incubated on ice for 1 h and then digested using a sonifier. Upon centrifugation (30 min, 21000 g), the supernatants were used for test reactions.

Example 5: Testing of P450 Candidates for DA Hydroxylation

[0091] Reaction mixture: [0092] 10-80 ?l 100 mM NADH (redox cofactor) [0093] 250 ?l 1 M Tris-HCl pH 7.5 [0094] 17.5 ?l glycerin (50%) [0095] 100 ?l 50 mM deoxycholic acid solution pH 8.5 (final 10 mM) [0096] 50 ?l E. coli lysate P450/PtR/Ptx (see Example 4) [0097] 17.5-87.5 ?l dH.sub.2O

[0098] The reactions were set up in 1.5 ml screw-top bottles and sealed with lids with aluminium foil. The foil was punctured in several places. It was gently shaken at 24? C. for 18 h. 200 ?l of the reaction batch was diluted with 600 ?l acetonitrile/5 ?l H.sub.3PO.sub.4 (50%) and incubated at 550? C. for 15 minutes. Subsequently, the samples were centrifuged at 20817 rcf for 5 minutes and analyzed using HPLC/DAD (e.g., Agilent 1200 series; [0099] column: Merck Purospher STAR RP-18e 125?4 mm, 5 ?m; [0100] flow rate: 1.5 ml/min, gradient H.sub.2O +H.sub.3PO.sub.4 (pH=2.6)/acetonitrile). One of the examined candidates (P450_c866) was able to hydroxylate deoxycholic acid to ursocholic acid. The deoxycholic acid used was converted in the process (see the following table). The identity of the product ursocholic acid was verified by GC/MS analysis and by 2D NMR.

TABLE-US-00006 Redox cofactor [?l] ursocholic acid [?g/ml] conversion [%] 10 30 3.4 30 48 5.3 50 57 6.2 80 110 11.6

[0101] In this example, a redox cofactor (NADH) is oxidized by the P450/Ptx/PtR reaction.