WO2018036982A1 - Chemical-biocatalytic method for producing ursodeoxycholic acid - Google Patents

Abstract

The invention relates to a further chemical-biocatalytic method for producing ursodeoxycholic acid from chenodeoxycholic acid after chemical oxidation to 3,7-diketo-ursodeoxycholic acid and after subsequent reaction with 7 beta-hydroxysteroid dehydrogenase from C. aerofaciens and 3 alpha-hydroxysteroid dehydrogenase from C. testosteroni.

Description

 Chemical-biocatalytic process for the production of ursodeoxycholic acid

The invention relates to an improved chemical-biocatalytic process for the production of ursodeoxycholic acid starting from chenodeoxycholic acid

Background of the invention:

 Bile acids are biomolecules required for the digestion and absorption of fats, fatty acids and hydrophobic vitamins. A bile acid that is found in low levels in humans is ursodeoxycholic acid (referred to as UDCA or UDCS). She has now gained great therapeutic importance in the dissolution of cholesterol-containing gallstones.

 For the medicinal treatment of gallstone disease u. a. the active substances ursodeoxycholic acid (UDCS or UDCA) and the associated diastereomer chenodeoxycholic acid (CDCS or CDCA). Both compounds differ only in the configuration of the hydroxyl group at C atom 7 (UDCS: ß configuration, CDCS: a configuration).

 For the production of UDCA, the prior art describes various processes which are carried out purely chemically or consist of a combination of chemical and enzymatic process steps. The starting point is cholic acid (CS or CA) or cholic acid-derived CDCA.

 An important precursor for the synthesis of UDCA is 12-ketodesodeoxycholic acid (12-keto-UDCA), which can be converted into UDCA by Wolff-Kishner reduction. A literature-described route for the synthesis of 12-ketoursodeoxycholic acid starts from cholic acid (3a, 7a, 12a-trihydroxy-5β-cholanic acid) catalyzed by two oxidative steps catalyzed by 7a and 12a HSDHs and a reductive step a 7ß-HSDH, can be prepared (Bovara R et al. (1996) A new enzymatic route to the synthesis of 12-ketoursodeoxycholic acid. Biotechnol. Lett. 18: 305-308; Monti D et al (2009) One- pot multienzymatic synthesis of 12-ketodesodeoxycholic acid: Subtle cofactor specificities rule the reaction equilibria of five biocatalysts working in a row. Adv. Synth. Catal. 351: 1303-131 1).

Another route starts from 7-ketolithocholic acid (7-keto-LCA), which can be converted into UDCA by stereoselective reduction of the 7-keto group. This step too is advantageously carried out enzymatically catalyzed catalyzed by a 7β-HSDH (Higashi S et al. (1979) Conversion of 7-ketolithocholic acid to ursodeoxycholic acid by human intestinal anaerobic microorganisms: interchangeability of chenodeoxycholic acid and ursodeoxycholic acid Gastroenterology Japonica 14: 417-424; Liu L et al. (201 1) Identification, cloning, heterologous expression, and characterization of a NADPH-dependent 7β-hydroxysteroid dehydrogenase from Collinsella aerofaciens. Appl. Microbiol. Biotechnol. 90: 127-135)..

 Another favorable synthetic route starts from dehydrocholic acid (DHCA), which can be converted into 12-keto-UDCA by two reductive steps, and these two steps can be catalyzed by two stereoselective HSDHs (3α and 7β-HSDHs) (Carrea G et al (1992) Enzymatic synthesis of 12-ketoursodeoxycholic acid from dehydro- cholic acid in a membrane reactor Biotechnol. Lett 14: 1 131-1 135; Liu L et al. (2013) One-step synthesis of 12-ketoursodeoxycholic acid from dehydrocholic acid using a multiency- matic system, Appl. Microbiol. Biotechnol. 97: 633-639).

 The classically chemical method of UDCS production can be schematically illustrated as follows:

CS CS methyl ester 3, 7-diacetyl-C S-methyl ester

Ein gravierender Nachteil ist unter anderem folgender: da die chemische Oxidation nicht selektiv ist, müssen die Carboxy-Gruppe und die 3a und 7a-Hydroxy-Gruppe durch Veresterung geschützt werden. 12-keto-3,7-diacetyl-CS-methylester

One serious drawback is as follows: since the chemical oxidation is not selective, the carboxy group and the 3a and 7a-hydroxy groups must be protected by esterification.

 An alternative chemical / enzymatic method based on the use of the enzyme 12a-hydroxysteroid dehydrogenase (12a-HSDH) can be represented as follows and is e.g. described in WO 2009/1 18176 of the present applicant.

12-keto-CDCS 7-keto-LCS UDCS

 The 12a-HSDH oxidizes CS selectively to 12-keto-CDCS. The two contactor steps required by the classical chemical method are eliminated.

 Furthermore, Monti, D., et al., (One-Pot Multienzymatic Synthesis of 12-Ketoursodeoxycholic Acid: Subtle Cofactor Specificities Rule the Reaction Equilibria of Five Biocatalysts Working in a Row., Advanced Synthesis & Catalysis, 2009) provides an alternative en- enzymatic-chemical process described schematically as follows:

CS »Γ 7,12-diketo-LCS

Die CS wird zuerst von 7a-HSDH aus Bacteroides fragilis ATCC 25285 (Zhu, D., et al., Enzymatic enantioselective reduction of -ketoesters by a thermostable 7 -hydroxysteroid dehydrogenase from Bacteroides fragilis. Tetrahedron, 2006. 62(18): p. 4535-4539) und 12a-HSDH zu 7,12-Diketo-LCS oxidiert. Diese beiden Enzyme sind jeweils NADH abhängig. Nach der Reduktion durch 7ß-HSDH (NADPH abhängig) aus Clostridium absonum ATCC 27555 (DSM 599) (MacDonald, I.A. and P.D. Roach, Bile induction of 7 alpha- and 7 beta- hydroxysteroid dehydrogenases in Clostridium absonum. Biochim Biophys Acta, 1981. 665(2): p. 262-9) entsteht 12-Keto-UDCS. Durch Wolff-Kishner-Reduktion wird das Endprodukt erhalten.

The CS is first extracted from 7a-HSDH from Bacteroides fragilis ATCC 25285 (Zhu, D., et al., Enzymatic enantioselective reduction of -ketoesters by a thermostable 7-hydroxysteroid dehydrogenase from Bacteroides fragilis, Tetrahedron, 2006. 62 (18): p 4535-4539) and 12a-HSDH to 7,12-diketo-LCS. These two enzymes each depend on NADH. After reduction by 7β-HSDH (NADPH dependent) from Clostridium absonum ATCC 27555 (DSM 599) (MacDonald, IA and PD Roach, Bile induction of 7 alpha- and 7-hydroxysteroid dehydrogenases in Clostridium absumum, Biochim Biophys Acta, 1981. 665 (2): p. 262-9) results in 12-keto-UDCS. By Wolff-Kishner reduction, the final product is obtained.

 As 7ß-HSDH, the enzyme from C. aerofaciens has been found to be well suited. In the meantime, the gene sequence of this enzyme from C. aerofaciens is known, so that on the one hand the enzyme can be made recombinantly available after cloning, on the other hand it is possible to generate mutants of this enzyme with methods of protein engineering and thus possibly find more advantageous enzyme variants. A 7β-HSDH from strain Collinsella aerofaciens ATCC 25986 (DSM 3979, former Eubacterium aerofaciens) ^^ described by Hirano and Masuda in 1982 (Hirano, S. and N. Masuda, Characterization of NADP-dependent 7-beta-hydroxysteroid dehydrogenases from Pep tostreptococcus productus and Eubacterium aerofaciens, Appl Environ Microbiol, 1982. 43 (5): pp. 1057-63). Sequence information was not disclosed for this enzyme. The molecular weight determined by gel filtration was 45,000 Da (see Hirano, page 1059, left column). Furthermore, the reduction of the 7-oxo group to the 7β-hydroxy group could not be observed for the enzyme there (see Hirano, page 1061, discussion 1st paragraph), the person skilled in the art thus recognizes that the enzyme described by Hirano et al Catalysis of the reduction of dehydrocholic acid (referred to herein as DHCS or DHCA) in the 7- position to 3,12-diketo-7β-CS.

 Applicant's International Patent Application WO201 1/064404 describes a novel 7β-HSDH from Collinsella aerofaciens ATCC 25986 which has, inter alia, a molecular weight (on SDS gel electrophoresis) of about 28-32 kDa, a molecular weight (on gel filtration, under non-denaturing Conditions, especially without SDS): from about 53 to 60 kDa and capable of stereoselective reduction of the 7-carbonyl group of 7-keto LCS to a 7β-hydroxy group.

 In addition, a method for producing UDCS is provided in WO201 1/064404, which is schematically represented as follows:

DHCS 12-keto-UDCS

The oxidation of CS takes place in a classical chemical way in a simple way. se. The DHCS is reduced by enzyme pair 7ß-HSDH and 3a-HSDH one by one or in a pot to 12-keto-UDCS. Combined with the Wolff-Kishner reduction, UDCS can thus be synthesized from CS in just three steps. While the 7ß-HSDH is dependent on the cofactor NADPH, the 3a-HSDH requires the cofactor NADH. The availability of enzyme pairs with dependency on the same cofactor or extended dependency (eg, of the cofactors NADH and NADPH) would be advantageous because it could simplify cofactor regeneration.

 WO 2012/080504 describes novel mutants of 7 .beta.-HSDH from C. aerofaciens in the sequence region of amino acid residues 36 to 42 of the C. aerofaciens sequence (SEQ ID NO: 1 1), as well as biocatalytic processes for the production of UDCS, and in particular also novel ones Whole cell method wherein 7ß-HSDH and 3a-HSDH act together on the substrate. Other novel mutants of C. aerofaciens 7ß-HSDH are disclosed in WO2015 / 197698 and WO2016 / 016213 by the present applicant

 As 3a-HSDH, the enzyme from C. testosteroni has proven to be well suited. In the meantime, the gene sequence of this enzyme is known, so that on the one hand the enzyme can be made recombinantly available after cloning, on the other hand it is possible to generate mutants of this enzyme with methods of protein engineering and thus possibly find more advantageous enzyme variants.

 WO 2016/023933 describes novel mutants of the 3a-HSDH from C. testosteronii, in particular in the mutation positions R34, L68 P185, T188 of SEQ ID NO: 8 as well as C- and N-terminal His-tag mutants

 WO 201 1/147957 describes novel knock-out strains which are particularly suitable for the production of UDCA, since the undesired 7a-HSDH enzyme activity could be deliberately switched off.

 Hwang et al. mention in PLOS ONE (2013) 8, 5, e63594 specific single mutants (P185A, G or W; T188A, S or W) and double mutants (W173F / P185W and W173F / T188W) of the 3a-HSDH from C. testosteronii. These mutants possessing N-His tags are proposed for NAD-dependent oxidation of the substrate androsterone. Other substrates are not discussed.

 The object of the invention is to provide a novel process for the production of UDCA in high yield.

Summary of the invention:

Surprisingly, the above objects have been achieved by providing a two-stage process for preparing UDCA from CDCA as defined in the appended claims, wherein CDCA is first chemically converted to 3,7-diketo-UDCA and then biocatalytically by in the presence of 7.beta.-HSDH and 3a-HSDH is stereospecifically reduced in high yield to UDCA.

Brief Description:

 FIG. 1 shows the SDS gel of hydroxysteroid dehydrogenases after expression in shake flask fermentation in LB medium, differentiated into the soluble fraction (1 and 4) and into purified fraction (2 and 3). The soluble fraction is the cell-free crude extra kt. In each case 10 μg of protein were applied. The size of the 3a-HSDH is about 26 kDa, that of the 7ß-HSDH about 29 kDa. 1/2 = 7β HSDH (C); 3/4 = 3a-HSDH (N); M = marker (Thermo scientific prestained ladder).

 FIG. 2 shows the Michaelis-Menten kinetics of 3a-HSDH for the substrate 3,7-diketo

UDCA (Figure A), for the cofactor NADH (Figure B). and for the intermediate 3-keto-UDCA (Figure C). The concentration range for 3,7-diketo-UDCA or 3-keto-UDCA is between 10 μΜ and 10 mM. For the coenzyme NADH, the concentration range was 10 to 300 μΜ. The enzyme was purified and used via an N-terminal hexahistidine residue.

 Figure 3 shows the Michaelis-Menten kinetics of 7β-HSDH for the substrate 3,7-diketo-UDCA (panel A), for the cofactor NADPH (panel B, with 3,7-diketo-UDCA as co-substrate); Picture D with 7-KLCA). and for the intermediate compound 7-KLCA (Figure C). The concentration range for 3,7-diketo-UDCA is between 10 μηη and 15 mM or 7-KLCA is between 10 μΜ and 10 mM. For the coenzyme NADPH, the concentration range was 10 to 300 μΜ. The enzyme was purified via a C-terminal hexahistidine residue and used.

 Figure 4 shows the conversion of 3,7-diketo-UDCA to UDCA after 16 hours at different pH values.

 Figure 5 shows the reaction with 40 mM 3,7-diketo-UDCA and the whole cell biocatalyst £. coli DB03.

 Figure 6 shows the reaction with 40 mM 3,7-diketo-UDCA and the whole cell biocatalyst £. coli DB02.

FIG. 7 shows a complete reaction scheme of a preferred variant of the inventive chemical / enzymatic process for the production of UDCA from CDCA, wherein the cofactor regeneration takes place by means of a GDH containing both NAD ⁺ and NADP ⁺ in the enzymatic oxidation of glucose to glucono -1, 5-lactone with regeneration of spent NADH or NADPH used.

Special embodiments of the invention The invention particularly relates to the following specific embodiments:

1 . Process for the preparation of a ursodeoxycholic acid (UDCA) of the formula (1)

R is alkyl, H, an alkali metal ion or N (R ³ ) ₄ ⁺ , in which the radicals R ^{3 are} identical or different and are H or alkyl, or where the group -C0 ₂ R by an acid amide group of the formula -ONR ¹ R ^{2 is} replaced, in which R ¹ and R ² independently of one another are an alkyl radical, and R is in particular H,

where you are

a) a chenodeoxycholic acid (CDCA) of the formula (2)

worin R oder die Gruppe -C0₂R die oben angegebenen Bedeutungen besitzen,

wherein R or the group -C0 ₂ R have the meanings given above,

to a 3,7-diketo-ursodeoxycholic acid (3,7-diketo -UDCA) of the formula (3)

wherein R or the group -C0 ₂ R have the meanings given above, chemically oxidized; and optionally purifying the formed 3,7-deco-UDCA, b) the 3,7-diketo-UDCA of formula (3) formed in step a) in the presence of at least one 7β-hydroxysteroid dehydrogenase (7β-HSDH) and in the presence of at least one 3α-hydroxysteroid dehydrogenase (3a-HSDH) to form the UDCA compound of above formula (1) enzymatically reduced, and optionally the reaction product further purified.

2. The method of embodiment 1, wherein in step a) subjected to a chemical oxidation tion, wherein the oxidizing agent used for the oxidation of a secondary hydroxyl group is capable. 3. Process according to embodiment 2, wherein the oxidation step is selected under a SWERN oxidation and a TEMPO oxidation and in particular an oxidation by means of sodium hypochlorite.

4. The method according to any one of the preceding embodiments, wherein step b) in the presence and consumption of reduction equivalents (NADH and / or

NADPH).

5. The method according to any one of the preceding embodiments, wherein the 3a-HSDH and 7ß-HSDH used in step b) have the same or different cofactor specificity.

6. The method according to any one of the preceding embodiments, wherein one couples stage b) for cofactor regeneration with at least one further, regenerating the spent regeneration enzyme.

7. The method of embodiment 6, wherein spent NADPH is regenerated by coupling with an NADPH regenerating enzyme, which is especially selected from NADPH dehydrogenases, alcohol dehydrogenases (ADH), and NADPH regenerating formate dehydrogenases (FDH) and an NADPH regenerating glucose dehydrogenase (GDH) and NADPH-regenerating glucose-6-phosphate dehydrogenases (G6PDH); and / or where spent NADH is regenerated by coupling with an NADH-regenerating enzyme, in particular being selected from NADH-dehydrogenases, NADH-regenerating formate dehydrogenases (FDH), NADH-regenerating alcohol dehydrogenases (ADH), NADH-regenerating glucose-6-phosphate dehydrogenases (G6PDH), NADH-regenerating phosphite dehydrogenases (PtDH) and NADH-regenerating glucose dehydrogenases (GDH); and / or wherein spent NADH and / or spent NADPH is regenerated by coupling with a cofactor regenerating enzyme selected from a NAD (P) H regenerating GDH.

8. Method according to embodiment 7, wherein spent NADH and / or spent NADPH is regenerated by coupling with a thermostable mutant regenerating both cofactors of a Bacillus subtilis GDH, in particular the E170K, Q262L double mutant (see Vazquez-Figueroa, Eduardo; Riggers, Javier, Bommarius, Andreas S., Chembiochem, 2007, 8, 2295-2301) according to SEQ ID NO: 14

9. The method of embodiment 7 or 8, wherein adding glucose to the reaction medium as a substrate for the GDH. 10. The process according to embodiment 9, wherein the reaction medium additionally NAD ⁺ and / or NADP ⁺ added to (additional) reduction equivalents for the enzymatic conversion to the 3,7-deco-UDCA compound of formula (3) according to step b ) to generate. 1 1. Method according to one of the preceding embodiments, wherein step b) with isolated or enriched enzymes (7ß-HSDH, the 3a-HSDH and optionally the enzyme required for cofactor regeneration) or with crude cell extract (containing the 7ß-HSDH, the 3a-HSDH and optionally the enzyme needed for cofactor regeneration) or with recombinant microorganisms which functionally express the 7ß-HSDH, the 3a-HSDH and optionally the enzyme required for cofactor regeneration, and preferably a 7ß-HSDH 3a-HSDH and the enzyme (s) required for cofactor regeneration.

12. The method according to embodiment 1 1, wherein the one uses a recombinant microorganism in which unwanted side activity of a 7a-HSDH is inhibited. 13. The method according to any one of the preceding embodiments, wherein the 7ß-

HSDH is selected from the following enzymes and enzyme mutants, wherein the non-mutated enzyme has an amino acid sequence according to SEQ ID NO: 1 1:

 WT = wild type; S = single mutation; D = double mutation; T = triple mutation; Q = quadruple mutation

 'Indication of the exclusively or preferably used cofactor (whereby the oxidized form of the corresponding cofactor is also included)

Included here are variants that are extended C- and / or N-terminal, with a His-tag sequence; and wherein the above mutants can additionally be mutated in each of the positions K44, R53, K61, R64.

14. Method according to one of the preceding embodiments, wherein the 3-HSDH is selected from the following enzymes and enzyme mutants, wherein the non-mu characterized enzyme has an amino acid sequence according to SEQ ID NO: 8.

 '' Specification of the exclusively or preferably used cofactor (wherein the oxidized form of the corresponding cofactor is also included)

WT = wild type; S = single mutation; D = double mutation

Included here are mutants that are C- and / or N-terminal, extended with a His-tag sequence.

15. The method according to any one of the preceding embodiments, wherein the following enzyme combinations are used

3a-HSDH 7ß-HSDH GDH

 C. testosteroni C. aerofaciens B. subtilis

 1 WT if necessary with N-His-tag WT if necessary with C-His-tag SEQ ID NO: 12 mutant

2 T188A, S, V, G or W WT optionally with C-His tag SEQ ID NO: 12 mutant 3a-HSDH 7ß-HSDH GDH

 C. testosteroni C. aerofaciens B. subtilis

 3 T188A, S, V, G or W G39S / R64E optionally with C-His tag SEQ ID NO: 12 mutant

4 WT optionally with N-His tag G39S / R64E optionally with C-His tag SEQ ID NO: 12 mutant

WT = wild type

In the above process, the reactions of step b) may be carried out in the presence of a recombinant microorganism (as further defined below), e.g. in the presence of whole cells of one or more different recombinant microorganisms, wherein the microorganism (s) carry the enzymes necessary for the reaction and cofactor regeneration in a manner described in more detail herein.

 The method step b) can be designed differently. Either both enzymes (7ß-HSDH mutant and 3a-HSDH) may be present concurrently (eg single-pot reaction with both isolated enzymes or one or more corresponding recombinant microorganisms are present expressing both enzymes) or the partial reactions may proceed in any order (first the 7β-HSDH mutant catalyzed reduction and then the 3a-HSDH catalyzed reduction, or first the 3a-HSDH catalyzed reduction and then the 7β-HSDH mutant-catalyzed reduction).

 Furthermore, step b) may be coupled to a cofactor regeneration system in which NADPH is regenerated by an NADPH regenerating enzyme such as, in particular, a GDH consuming glucose (particularly when employing a 7ß-HSDH and 3a-HSDH which both utilize NADPH); or coupled to a cofactor regeneration system in which spent NADH is regenerated by an enzyme regenerating NADH, such as, in particular, a GDH, ADH or FDH (particularly when using a 7ß-HSDH and 3a-HSDH both use NADH);

 Furthermore, step b) can be coupled to a cofactor regeneration system in which NADPH and NADH are regenerated by an enzyme which regenerates both NADPH and NADH, in particular a GDH with consumption of glucose (in particular if a 7ß-HSDH and 3a-HSDH are used will use the different cofactors.

16. Recombinant microorganism according to the definition in one of the embodiments 1 1 and 12.

17. bioreactor for carrying out a method according to one of the embodiments

1 to 15, in particular containing at least one of the enzymes used in step b). The present invention is not limited to the specific embodiments described herein. The person skilled in the art will be enabled by the teachings of the present invention to provide further embodiments of the invention without undue burden. For example, it can also generate additional enzyme mutants in a targeted manner and screen and optimize them for the desired property profile; or other suitable wild-type enzymes (7ß and 3a-HSDHs, FDHs, GDHs ADHs, etc.) and isolate according to the invention. Furthermore, depending on the profile of properties (in particular cofactor dependency) of the HSDHs used, in particular 7ß-HSDH and 3a-HSDH or mutants thereof, it can for example select suitable dehydrogenases useful for cofactor regeneration (GDH, FHD, ADH etc.) and mutants thereof, and distribute the selected enzymes to one or more expression constructs or vectors and, if necessary, generate one or more recombinant microorganisms which then enable a further optimized enzymatic or, in particular, whole-cell production of UDCA.

Further embodiments of the invention

 1 . General definitions and abbreviations used

 Unless otherwise specified, the term "7ß-HSDH" refers to a dehydrogenase enzyme which is capable of at least the stereospecific and / or regiospecific reduction of DHCS or 7,12-diketo-3a-CS (7,12-diketo). LCS) to 3,12-diketo-7ß-CS or 12-keto-UDCS, in particular with stoichiometric consumption of NADPH, and optionally catalyzes the corresponding inverse reaction, the enzyme may be a native or recombinantly produced enzyme Suitable detection methods are described, for example, in the following experimental section or are known from the literature (eg characterization of NADP-dependent 7beta-hydroxysteroid dehydrogenases from Peptostreptococcus productus and Eubacterium aerofoets) S. Hirano and N. Masuda, Appl Environ Microbiol., 1982). Enzymes of this activity are classified under EC number 1.1 .1.201 enzymes were e.g. isolatable from Colinseilea aerofaciens.

Unless otherwise specified, the term "3a-HSDH" refers to a dehydrogenase enzyme which comprises at least the stereospecific and / or regiospecific reduction of 3,12-diketo-7ß-CS or DHCS to 12-keto-UDCS or 7.12-diketo-3a-CS (7,12-diketo-LCS), in particular with stoichiometric consumption of NADH and / or NADPH, and optionally catalyzes the corresponding inverse reaction Suitable detection methods are described, for example, in the following experimental section or known from the literature. Suitable enzymes are available, for example, from Comanomonas testosteroni (ie Comamonas testosteroni) (eg ATCC1 1996). A NADPH-dependent 3a-HSDH is known, for example, from rodents and can also be used. (Cloning and sequencing of the cDNA for rat liver 3 alpha-hydroxysteroid / dihydrodiol dehydrogenase, JE Pawlowski, M. Huizinga and TM Penning, May 15, 1991 The Journal of Biological Chemistry, 266, 8820-8825). Enzymes of this activity are classified under EC number 1.1.1.50.

Unless otherwise specified, the term "GDH" refers to a dehydrogenase enzyme which comprises at least the oxidation of β-D-glucose to D-glucono-1,5-lactone with stoichiometric consumption of NAD ⁺ and / or NADP ⁺ and optionally catalysed the corresponding reverse reaction. Suitable enzymes are, for example megaterium obtainable from Bacillus subtilis or Bacillus. enzymes of this type are classified under the EC number 1 .1 .1 .47.

Unless otherwise specified, the term "FDH" refers to a dehydrogenase enzyme which comprises at least the oxidation of formic acid (or corresponding formate salts) to carbon dioxide with stoichiometric consumption of NAD ⁺ and / or NADP ⁺ , and Suitable detection methods are described, for example, in the following experimental section or are known from the literature Suitable enzymes are obtainable, for example, from Candida boidinii, Pseudomonas sp or Mycobacterium vaccae Enzymes of this activity are listed under EC number 1.2.1 .2 classified.

 A "pure form" or a "pure" or "substantially pure" enzyme is understood according to the invention to mean an enzyme having a purity of more than 80, preferably more than 90, in particular more than 95, and in particular more than 99% by weight. , based on the total protein content, determined by means of conventional protein detection methods, such as, for example, the biuret method or the protein detection according to Lowry et al. (see description in RK Scopes, Protein Purification, Springer Verlag, New York, Heidelberg, Berlin (1982) ).

A "redox equivalent" is understood to mean a low molecular weight organic compound which can be used as an electron donor or electron acceptor, for example nicotinamide derivatives such as NAD ⁺ and NADH ⁺ or their reduced forms NADH or NADPH. "Redox equivalent" and "cofactor" are used in the context Thus, a "cofactor" within the meaning of the invention can also be described as an "redox-capable cofactor", ie as a cofactor, which may be present in reduced and oxidized form.

A "spent" cofactor is understood to mean that reduced or oxidized form of the cofactor which is converted into the corresponding oxidized or reduced form in the course of a given reduction or oxidation reaction of a substrate reduced cofactor Form again transferred back into the reduced or oxidized starting form, so that it is again available for the implementation of the substrate.

 In the context of the present invention, a "modified cofactor use" is understood to mean a qualitative or quantitative change in comparison to a reference. In particular, altered cofactor utilization can be observed by making amino acid sequence mutations. This change is then detectable in comparison to the non-mutated starting enzyme. In this case, the activity with respect to a particular cofactor can be increased or decreased by making a mutation or be completely suppressed. However, an altered use of cofactor also includes changes such that, instead of a specificity for a single cofactor, at least one further second cofactor different from the first cofactor can be used (ie an extended cofactor utilization is available) existing ability to use two different cofactors are modified so that specificity is only increased for one of these cofactors or reduced for one of these cofactors or completely abolished. For example, an enzyme that is dependent on the cofactor NAD (NADH) may now be dependent on NAD (NADH) or the cofactor NADP (NADPH) due to a change in cofactor use, or may have the initial dependence on NAD (NADH) completely converted into a dependence on NADP (NADPH) and vice versa.

 The terms "NADVNADH-dependency" and "NADPVNADPH-dependency" are according to the invention, unless otherwise defined, to be interpreted broadly. These terms encompass both "specific" dependencies, i.e., solely dependence on NAD7NADH and NADP7NADPH, as well as the dependence of the enzymes of the invention on both cofactors, i.e., dependence on NAD7NADH and NADP7NADPH.

 The same applies to the terms "NAD7NADH-accepting" or

"NADPVNADPH-accepting."

 The terms "NAD7NADH-regenerating" or "NADP7NADPH-regenerating" are according to the invention, unless otherwise defined, to be interpreted broadly. These terms include both "specific", i.e., exclusive, ability to regenerate spent cofactor NAD7NADH and NADP7NADPH, as well as the ability to regenerate both cofactors, i.e., NAD7NADH and NADP7NADPH.

 In particular, "proteinogenic" amino acids include (one-letter code): G, A, V, L, I, F, P, M, W, S, T, C, Y, N, Q, D, E, K, R and H.

According to the invention, "immobilization" is understood as meaning the covalent or noncovalent binding of a biocatalyst used according to the invention, such as, for example, a 7β-HSDH to a solid carrier material, ie essentially insoluble in the surrounding liquid medium the recombinant microorganisms used according to the invention may be immobilized with the aid of such carriers.

 A "substrate inhibition reduced in comparison with the non-mutated enzyme" means that the substrate inhibition observed for the unmutated enzyme for a particular substrate is no longer observable, ie essentially no longer measurable, or only starts at a higher substrate concentration , ie the Kr value is increased.

 N- or C-terminal extensions according to the invention comprise about 15 to 25 proteinogenic amino acid residues which, optionally reversible, e.g. via a protease interface attached to a terminal amino acid residue. Preferred residues include so-called "His-tags" known in the art with, for example, 3, 6 or 9 consecutive histidine residues in the extension sequence.

 According to the invention, a "cholic acid compound" is understood as meaning compounds having the basic carbon structure, in particular the steroid structure of cholic acid and the presence of keto and / or hydroxy or acyloxy groups in the ring position 7 and optionally the ring positions 3 and / or 12.

 Under a compound of a special type, e.g. In particular, a "cholic acid compound" or a "ursodeoxycholic acid compound" is also understood as meaning derivatives of the parent parent compound (such as cholic acid or ursodeoxycholic acid).

Such derivatives include "salts" such as alkali metal salts such as lithium, sodium and potassium salts of the compounds, as well as ammonium salts wherein the ammonium salt comprises the NH ₄ ⁺ salt or ammonium salts wherein at least one hydrogen atom is represented by a CrC ₆ alkyl radical may be replaced. Typical alkyl groups are, in particular -C ₄ alkyl radicals such as methyl, ethyl, n- or i-propyl, n-, sec- or tert-butyl, and also n-pentyl and n-hexyl, and the a or multi-branched analogs thereof

"Alkyl esters" of compounds according to the invention are in particular lower alkyl esters, such as, for example, C 1 -C ₆ -alkyl esters, non-limiting examples being methyl, ethyl, n- or i-propyl, n-, sec- or tert-butyl ester, or longer chain esters such as n-pentyl and n-hexyl esters and the mono- or polysubstituted analogs thereof.

"Amides" are, in particular, reaction products of acids according to the invention with ammonia or primary or secondary monoamines. "Such amines are, for example, mono- or di-C ₁ -C ₆ -alkyl-monoamines, where the alkyl radicals independently of one another may optionally be further substituted, for example by carboxy- , Hydroxy, halogen (such as F, Cl, Br, J), nitro and sulfonate groups.

"Acyl groups" according to the invention are in particular nonaromatic groups having 2 to 4 carbon atoms, such as acetyl, propionyl and butyryl, and aromatic groups with optionally substituted mononuclear aromatic ring, suitable substituents being, for example, selected from hydroxy, halogen (such as F, Cl, Br , J) -, Nitro- and Ci-C ₆ alkyl groups, such as benzoyl or toluoyl.

 The hydroxysteroid compounds used according to the invention, such as e.g. Cholic acid, ursodeoxycholic acid, 12-keto-chenodeoxycholic acid, chenodeoxycholic acid and 7-keto-lithocholic acid can be used in stereoisomerically pure form or in admixture with other stereoisomers in the process of the invention or obtained therefrom. Preferably, however, the compounds used or the compounds prepared are used or isolated in essentially stereoisomerically pure form.

The following table summarizes the structural formulas, their chemical names, and the abbreviations used for essential chemical compounds in tabular form:

2. proteins

 The present invention is not limited to the specifically disclosed proteins or enzymes having 7β-HSDH, FDH, ADH-GDH or 3a-HSDH activity or their mutants, but rather also extends to functional equivalents thereof.

 "Functional equivalents" or analogues of the specifically disclosed enzymes are, within the scope of the present invention, various polypeptides which furthermore possess the desired biological activity, such as, for example, HSH activity.

 Thus, for example, "functional equivalents" are understood to mean enzymes which, in the test used for 7ß-HSDH, ADH, FDH, GDH or 3a-HSDH activity, increase by at least 1%, such as at least 10% or 20%, for example. such as at least 50% or 75% or 90% higher or lower activity of a parent enzyme comprising an amino acid sequence as defined herein.

 Functional equivalents are also preferably stable between pH 4 to 1 1 and advantageously have a pH optimum in a range of pH 6 to 10, in particular 8.5 to 9.5, and a temperature optimum in the range of 15 ° C to 80 ° C or 20 ° C to 70 ° C, such as about 45 to 60 ° C or about 50 to 55 ° C.

 The 7β-HSDH activity can be detected by various known tests. Without being limited to this, let a test be performed using a reference substrate, such as a reference substrate. CS or DHCS, under standardized conditions as defined in the experimental section.

Tests to determine ADH, FDH, GDH, 7ß-HSDH or 3a-HSDH activity are also known per se. According to the invention, "functional equivalents" are understood to mean, in particular, "mutants" which, in at least one sequence position of the abovementioned amino acid sequences, have a different amino acid than the one specifically mentioned but nevertheless possess one of the abovementioned biological activities. "Functional equivalents" thus include those represented by one or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, amino acid additions , Mutations, deletions and / or inversions, which changes can occur in any sequence position, as long as they lead to a mutant with the property profile according to the invention Functional equivalence is especially given when the reactivity patterns between mutants and unchanged polypeptide, ie, for example, the same substrates are reacted at different rates, examples of suitable amino acid substitutions are summarized in the following table:

Original rest Examples of substitution

 Ala Ser

 Arg Lys

 Asn Gin; His

 Asp Glu

 Cys Ser

 Gin Asn

 Glu Asp

 Gly Pro

 His Asn; gin

 all leuu; Val

 Hell! Val

 Lys Arg; Gin; Glu

 Met Leu; lle

 Phe Met; Leu; Tyr

 Ser Thr

 Thr Ser

 Trp Tyr

 Tyr Trp; Phe

 Val lle; Leu

"Functional equivalents" in the above sense are also "precursors" of the described polypeptides as well as "functional derivatives" and "salts" of the polypeptides.

 "Precursors" are natural or synthetic precursors of the polypeptides with or without the desired biological activity.

Salts are understood as meaning both salts of carboxyl groups and acid addition salts of amino groups of the protein molecules according to the invention. inorganic salts such as sodium, calcium, ammonium, iron and zinc salts, as well as salts with organic bases such as amines such as triethanolamine, arginine, lysine, piperidine and the like. Acid addition salts, such as, for example, salts with mineral acids, such as hydrochloric acid or sulfuric acid, and salts with organic acids, such as acetic acid and oxalic acid, are likewise provided by the invention.

 "Functional derivatives" of polypeptides of the invention may also be prepared at functional amino acid side groups or at their N- or C-terminal end by known techniques Such derivatives include, for example, aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups obtainable by reaction with ammonia or with a primary or secondary amine; N-acyl derivatives of free amino groups prepared by reaction with acyl groups; or O-acyl derivatives of free hydroxyl groups prepared by reaction with acyl groups.

 Of course, "functional equivalents" also include polypeptides that are accessible from other organisms, as well as naturally occurring variants. For example, it is possible to determine regions of homologous sequence regions by sequence comparison and to determine equivalent enzymes on the basis of the specific requirements of the invention.

 "Functional equivalents" also include fragments, preferably single domains or sequence motifs, of the polypeptides of the invention having, for example, the desired biological function.

 "Functional equivalents" are also fusion proteins having one of the above-mentioned polypeptide sequences or functional equivalents derived therefrom and at least one further functionally distinct heterologous sequence in low C-terminal functional linkage (ie, with no substantial substantial functional impairment of the fusion protein moieties) Examples of such heterologous sequences are, for example, signal peptides, histidine anchors or enzymes.

 Homologs to the specifically disclosed proteins encompassed by the invention include at least 60%, preferably at least 75%, in particular at least 85%, such as 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, homology (or identity) to one of the specifically disclosed amino acid sequences calculated according to the algorithm of Pearson and Lipman, Proc Natl Acad, S. (USA) 85 (8), 1988, 2444-2448 Homology or identity of a homologous polypeptide of the invention means, in particular, percent identity of the amino acid residues relative to the total length of one of the amino acid sequences specifically described herein.

 The percent identity values can also be determined using BLAST alignments, algorithm blastp (protein-protein BLAST), or by applying the Clustal settings below.

In the case of possible protein glycosylation, "functional" onal equivalents "proteins of the type described above in deglycosylated or glycosylated form as well as modified forms obtainable by altering the glycosylation pattern.

 Homologs of the proteins or polypeptides of the invention may be generated by mutagenesis, e.g. by point mutation, extension or shortening of the protein.

 Homologs of the proteins of the invention can be prepared by screening combinatorial libraries of mutants, such as e.g. Shortening mutants, to be identified. For example, a variegated library of protein variants can be generated by combinatorial mutagenesis at the nucleic acid level, e.g. by enzymatic ligation of a mixture of synthetic oligonucleotides. There are a variety of methods that can be used to prepare libraries of potential homologs from a degenerate oligonucleotide sequence. The chemical synthesis of a degenerate gene sequence can be performed in a DNA synthesizer, and the synthetic gene can then be ligated into a suitable expression vector. The use of a degenerate gene set allows for the provision of all sequences in a mixture that encode the desired set of potential protein sequences. Methods of synthesizing degenerate oligonucleotides are known to those skilled in the art (eg, Narang, SA (1983) Tetrahedron 39: 3; Itakura et al. (1984) Annu. Rev. Biochem. 53: 323; Itakura et al., (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acids Res. 1 1: 477).

 Several techniques for screening gene products of combinatorial libraries made by point mutations or truncation and for screening cDNA libraries for gene products having a selected property are known in the art. These techniques can be adapted to the rapid screening of gene libraries generated by combinatorial mutagenesis of homologs of the invention. The most commonly used techniques for screening large libraries that are subject to high throughput analysis include cloning the library into replicable expression vectors, transforming the appropriate cells with the resulting vector library, and expressing the combinatorial genes under conditions that include detection of the desired activity facilitates the isolation of the vector encoding the gene whose product has been detected. Recursive ensemble mutagenesis (REM), a technique that increases the frequency of functional mutants in the banks, can be used in combination with the screening assays to identify homologs (Arkin and Yourvan (1992) PNAS 89: 781 1 -7815; Delgrave et al., (1993) Protein Engineering 6 (3): 327-331).

2.1 7ß-HSDHs: 2.1 .1 The invention further encompasses the use of the wild type 7ß-HSDH from Collinsella aerofaciens ATCC 25986, as described in WO2011 / 064404 of the Applicant, to which reference is hereby expressly made.

 This 7β-HSDH obtainable from Collinsella aerofaciens DSM 3979 is especially characterized by at least one of the following properties, e.g. 2, 3, 4, 5, 6 or 7 or all such properties, characterized:

 a) Molecular weight (SDS gel electrophoresis): about 28-32 kDa, especially about 29-31 kDa or about 30 kDa;

 b) Molecular weight (gel filtration, under non-denaturing conditions, such as in particular without SDS): about 53 to 60 kDa, in particular about 55 to 57 kDa, such as 56.1 kDa. This demonstrates the dimeric nature of the 7ß-HSDH from Collinsella aerofaciens DSM 3979;

 c) stereoselective reduction of the 7-carbonyl group of 7-keto-LCS into a 7ß-hydroxy group;

 d) pH optimum for the oxidation of UDCS in the range of pH 8.5 to 10.5, in particular 9 to 10;

 e) pH optimum for the reduction of DHCS and 7-keto-LCS in the range of pH 3.5 to 6.5, in particular at pH 4 to 6;

 f) at least one kinetic parameter from the following table for at least one of the substrates / cofactors mentioned therein; in the range of ± 20%, in particular ± 10%,

± 5%, ± 3% ± 2% or ± 1% by the specific value given in the following table.

 could not be determined due to the very low activity

 1 U = 1μη "ΐοΙ / η" ΐίη g) Phylogenetic sequence relatedness of the prokaryotic 7ß-HSDH from Collinsella aerofaciens DSM 3979 with the animal 1 1 ß-HSDH subgroup, including Cavia porcellus, Homo sapiens and Mus musulus, related.

For example, this 7ß-HSDH exhibits the following properties or combinations of properties; a); b); a) and b); a) and / or b) and c); a) and / or b) and c) and d); a) and / or b) and c) and d) and e); a) and / or b) and c) and d) and e) and f).

 Such a 7β-HSDH or functional equivalent derived therefrom is further characterized by

 a. the stereospecific reduction of a 7-keto steroid to the corresponding 7β-hydroxysteroid, and / or

 b. the regiospecific hydroxylation of a ketosteroid comprising a 7-position keto group and at least one additional keto group on the steroid backbone catalyzed to the corresponding 7β-hydroxysteroid, in particular by dehydrocholic acid (DHCS) 7-position to the corresponding 3,12-diketo-7β-cholanic acid; and eg NADPH is dependent.

Such a 7ß-HSDH in particular has an amino acid sequence according to SEQ ID NO: 1 1 (Accession NO: ZP_01773061) or a sequence derived therefrom with an identity of at least 60%, such as. at least 65, 70, 75, 80, 85, or 90, e.g. at least 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.5% to this sequence; optionally additionally characterized by one of the following properties or combinations of properties; a); b); a) and b); a) and / or b) and c); a) and / or b) and c) and d); a) and / or b) and c) and d) and e); a) and / or b) and c) and d) and e) and f) as defined above.

2.1 .2 The invention further encompasses the application of 7β-HSDH mutants as described in WO2012 / 080504 of the Applicant, to which reference is hereby expressly made.

In particular, reference should be made to the following specific embodiments:

(1) A 7ß-HSDH mutant which catalyzes at least the stereospecific enzymatic reduction of a 7-keto steroid to the corresponding 7-hydroxysteroid, wherein the mutant compared to the non-mutated enzyme, in particular an enzyme comprising SEQ ID NO: 1 1 and / or at least the sequence motif VMVGRRE according to position 36 to 42 thereof, has a reduced substrate inhibition (in particular for the 7-ketosteroid substrate); and / or an altered cofactor use (eg increased, altered specificity with respect to a cofactor (in particular NADH or NADPH) or an extended dependence, ie use of an additional, previously unused cofactor), in particular such a mutant which does not show any substrate inhibition or that it has a value for the 7-ketosteroid substrate, in particular for dehydrocholic acid (DHCS), in the range of> 10 mM, such as 1 to 200 mM, 12 to 150 mM, 15 to 100 mM.

(2) A mutant according to (1), wherein the specific activity (U / mg) in the presence of the cofactor NADPH in comparison to the non-mutated enzyme by at least 1, 5 or 10%, but in particular at least one-fold, in particular by 2 to 10 times increased or decreased; or substantially lacking and being replaced by the use of NADH, or having extended use of NADPH and HADH).

(3) A 7ß-HSDH mutant which catalyzes at least the stereospecific enzymatic reduction of a 7-keto steroid to the corresponding 7-hydroxysteroid, optionally according to one of the preceding embodiments, wherein the mutant has at least one mutation in the amino acid sequence according to SEQ ID NO: 1 1 or an amino acid sequence derived therefrom having at least 60% sequence identity, such as at least 65, 70, 75, 80, 85, or 90, e.g. at least 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.5% to this sequence.

(4) A 7ß-HSDH mutant which catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7-hydroxysteroid, optionally according to one of the preceding embodiments, wherein the mutant at least one mutation in the sequence motif VMVGRRE according to position 36 to 42 of SEQ ID NO: 1 1 or in the corresponding sequence motif of an amino acid sequence derived therefrom having at least 60% sequence identity, such as at least 65, 70, 75, 80, 85, or 90, e.g. at least 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.5% to this sequence.

(5) mutant according to (3) or (4), selected from

a) the simple mutants G39Xi and R40X _2. ,

b) o- the double mutants (G39Xi, R40X _{2), (2} R40X, R41X ₃₎ and (G39Xi, R41X ₃₎

c) the triple mutant (G39Xi, R40X ₂ , R41X ₃ ),

 (in each case based on SEQ ID NO: 1 1)

wherein Xi, X ₂ and X _{3 are} each independently of one another any, in particular any, the substrate inhibition-reducing and / or the Kofaktornutzung or cofactor dependence modifying, different from G or R, in particular natural amino acid;

 or

d) the corresponding single, double or triple mutants of one of SEQ ID NO: 1 1 deduced amino acid sequence having at least 60% sequence identity, such as at least 65, 70, 75, 80, 85, or 90, such as at least 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.5% to this sequence. Examples of suitable single mutants include: G39A, G39S, G39D, G39V, G39T, G39P, G39N, G39E, G39Q, G39H, G39R, G39K and G39W, and R40D, R40E, R40I, R40V, R40L, R40G, R40A.

Examples of suitable double mutants include:

Two combinations of the above G39Xi and R40X ₂ mutants, wherein Xi is in particular D or E and / or X _{2 is} any amino acid, in particular proteinogenic amino acid, in particular an amino acid having an aliphatic side chain, such as (G39D, R40I), (G39D , R40L), (G39D, R40V); and the analog double mutants with G39E instead of G39D; such as

Two combinations of the above G39XrMutanten R40X or R41X ₂ mutants with ₃ mutants, wherein X ₃ is any, in particular a substrate inhibition, the reducing and / or the Kofaktornutzung Kofaktorabhängigkeit or modifying different from R in particular natural amino acid, such as the type (G39Xi, R41 X ₃ ) or (R40X ₂ , R41X ₃ ), eg with Xi = D or E; X ₂ = 1, L or V; X ₃ = N, I, L or V)

such as. (R40D, R41 I); (R40D, R41L); (R40D, R41V); (R40I, R41I); (R40V, R41I), (R40L, R41I).

oder E; R40X₂=I, L oder V; R41X₃= N, I, L oder V), wie z.B. (G39D,R40I,R41 N). Examples of suitable triple mutants include any triad combinations of the above single mutants G39Xi, R40X ₂ and R41X ₃ ; wherein Xi, X ₂ and X _{3 are} as defined above; but in particular wherein Xi is D, or E and / or X ₂ and X ₃ independently of one another represent an arbitrary amino acid, in particular a proteinogenic amino acid, such as, in particular, three-fold mutants of the type

or E; R40X ₂ = I, L or V; R41X ₃ = N, I, L or V), such as (G39D, R40I, R41 N).

Optionally, the mutants (1) to (5) additionally or alternatively, in particular additionally, at least one further substitution, such as 1, 2, 3 or 4 substitutions in the positions K44, R53, K61 and R64. In this case, these radicals can be replaced independently of one another by an arbitrary amino acid, in particular a proteinogenic amino acid, in particular a substitution such that the mutant thus produced has a reduced substrate inhibition (in particular for the 7-ketosteroid substrate); and / or altered cofactor use or cofactor dependency (eg, increased, altered cofactor specificity or extended dependency, that is, use of an additional, previously unused cofactor) as defined herein. 2.1 .3 The invention further encompasses the application of 7β-HSDH mutants as described in the Applicant's WO2015 / 197698, to which reference is hereby expressly made. In particular, reference should be made to the following specific embodiments:

(1) A 7ß-HSDH mutant which catalyzes at least the stereospecific enzymatic reduction of a 7-keto steroid to the corresponding 7-hydroxysteroid, the enzyme each having a mutation in the positions G39 and R40 of SEQ ID NO: 1 1 and, if appropriate in the positions R41 and / or optionally the position K44 of SEQ ID NO: 1 1 or in the respective corresponding sequence positions of an amino acid sequence derived therefrom having at least 80% sequence identity to SEQ ID NO: 1 1, such as at least 85, or 90, e.g. at least 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.5% to this sequence.

(2) 7ß-HSDH mutant according to (1) comprising the double mutation G39D / R40F.

(3) 7ß-HSDH mutant according to (1) or (2), comprising the triple mutation G39D / R40F / R41X-I, wherein Xi for any other amino acid residue, especially proteinogenic amino acid residue, in particular a NADH-specificity enhancing residue, in particular for K, Q, S or R and especially for K stands.

(4) 7ß-HSDH mutant according to (1) or (2)), comprising the four-fold mutation G39D / R40F / R41 Xi, / K44X ₂ , wherein Xi for any other amino acid residue, in particular proteinogenic amino acid residue, in particular a NADH Specificity increasing radical especially for K, Q, S or R and especially K, and X _{2 is} any other amino acid residue, in particular proteinogenic amino acid residue, in particular a NADH-specificity enhancing radical, in particular for G, N or Q, and especially G stands.

(5) 7ß-HSDH mutant according to (1) to (4), which shows the following property profile compared to 7ß-HSDH with SEQ ID NO: 1 1:

a. an increased specific activity (Vmax [U / mg]) for NADH in the enzymatic reduction of DHCA with NADH as cofactor; and / or b. an increased specific activity (Vmax [U / mg]) for NADH in the enzymatic reduction of 7-ketosteroids (especially bile salts with a keto group in position C7 of the steroid ring), with NADH as cofactor. 2.1.4 The invention further comprises the application of 7β-HSDH mutants as described in the Applicant's WO2016 / 016213, to which reference is hereby expressly made.

In particular, reference should be made to the following specific embodiments:

(1) 7ß-HSDH mutant which catalyzes at least the stereospecific enzymatic reduction of a 7-keto steroid to the corresponding 7-hydroxysteroid, wherein the enzyme is derived from an enzyme having the SEQ ID NO: 1 1, or an enzyme comprising this sequence, such as an enzyme comprising SEQ ID NO: 1 1 N-terminally extended by a histidine tag or histidine anchor sequence, and wherein the enzyme has a mutation in position 17 and / or 64 of SEQ ID NO: 1 1 (optionally N-terminal extended) or in the corresponding sequence positions of an amino acid sequence derived therefrom with at least 80%, such. At least e.g. 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.5% sequence identity, to SEQ ID NO: 1 1.

(2) 7ß-HSDH mutant catalyzing at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7-hydroxysteroid, and

an amino acid sequence modified by amino acid mutation in SEQ ID NO: 1 1 (optionally extended N-terminally), wherein the amino acid sequence mutation is selected from single or multiple mutations comprising:

b) T17X ₂

where Xi is an amino acid residue other than arginine (R), in particular any amino acid which increases specific activity and / or reduces substrate inhibition and / or modifies the utilization of cofactor or cofactor, in particular natural;

stands; and

and X ₂ for a proteinogenic amino acid residue other than threonine (T) is, in particular, any specific, particularly activity-enhancing, and / or substrate-inhibiting and / or cofactor-dependency-modifying, especially natural, amino acid;

wherein the mutated, ie altered amino acid sequence has a sequence identity to SEQ ID NO: 1 1 (optionally N-terminally extended) of 80% to less than 100%, such as 85, 90, 91, 92, 93, 94, 95, 96 , 97, 98, 99 or 99.5% sequence identity, preferably at least 85%, in particular at least 90%. (3) 7ß-HSDH according to (1) or (2), which additionally at least one mutation in the sequence motif VMVGRRE according to position 36 to 42, in particular position 39 of SEQ ID NO: 1 1 (optionally N-terminally extended) or in the corresponding Sequence motif of an amino acid sequence derived therefrom having at least 80% sequence identity, such as 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.5% sequence identity, to SEQ ID NO: 11 (optionally extended N-terminal)).

7ß-HSDH according to (3), which additionally has the amino acid sequence mutation c) G39X ₃ ,

wherein X _{3 stands} for a different from glycine (G), in particular proteinogenic amino acid residue, in particular any, specific activity-enhancing and / or the substrate inhibition-reducing and / or the Kofaktornutzung or cofactor dependency modifying, in particular natural amino acid stands.

7ß-HSDH according to one of the preceding embodiments, selected from a) the single mutant

T17X ₂ and the

 b) the double mutants

R64X ₁ / G39X ₃

 wherein

 Xi for E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I, Y, H or N, especially E, D, T, L, S, P or V stands;

X _{2 is} F, A, I, or S.

X _{3 represents} S, A, V, I, L, C, K, YF or R, in particular S or A.

Non-limiting examples of suitable single mutants include:

R64A, R64S, R64D, R64V, R64T, R64P, R64N, R64E, R64Q, R64H, R64RL, R64K, R64C, R64G, R64I, R64Y, R64F and R64W, and

T17F, T17A, T17I, T17S.

 Non-limiting examples of suitable double mutants include:

(G39S / R64E); (G39S / R64D); (G39S / R64T); (G39S / R64L); (G39S / R64S); (G39S / R64P); (G39S / R64V); (G39a / R64E); (G39a / R64D); (G39a / R64T); (G39a / R64S); (G39a / R64L); (G39a / R64P); (G39a / R64V); The above statements on single and double mutants apply in particular to SEQ ID NO: 11 and the N-terminally extended form thereof

(6) 7ß-HSDH according to one of the preceding embodiments, which shows at least one of the following properties or property profile compared to the non-mutated 7ß-HSDH with SEQ ID NO: 1 1 (optionally N-terminally extended):

 a) an increased specific activity (Vmax [U / mg]) for dehydrocholic acid (DHCA) in the enzymatic reduction of DHCA with NAD (P) H, in particular NADPH, as cofactor; whereby e.g. the specific activity (U / mg) in the presence of the cofactor NAD (P) H, in particular NADPH, in comparison to the non-mutated enzyme by at least 1, 5, 10, 50 or 100%, but in particular at least 1-fold, in particular is increased 2 to 100 times or 3 to 20 times or 5 to 10 times.

 b) an increased specific activity (Vmax [U / mg]) for NAD (P) H, in particular NADPH, in the enzymatic reduction of DHCA with NAD (P) H, in particular NADPH, as cofactor; whereby e.g. the specific activity (U / mg) in the presence of the cofactor NAD (P) H, in particular NADPH, in comparison to the non-mutated enzyme by at least 1, 5 or 10%, but in particular at least 1-fold, especially around 2 to 10 times increased

 c) decreased substrate inhibition by DHCA, e.g. with Ki values in the range of> 1 mM, e.g. at 1 to 200 mM, 2 to 150 mM, 2.5 to 100 mM; d) altered cofactor specificity with respect to NADH and NADPH, e.g. an extended specificity, ie use of an additional, previously unused cofactor, in particular NADPH;

 e) wherein these properties a) to d) can be present individually or in any desired combination.

Further specific embodiments relate to mutants of 7ß-HSDH with SEQ ID NO: 1 1 (optionally N-terminally extended) or a sequence derived therefrom with a degree of identity of at least 80% or at least 85%, in particular at least 90% to the wild-type sequence with at least one the above properties a), b), c), d) or e).

Further examples are:

For example, the simple mutants R64X-I in which X-1 is E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I, Y, H or N, in particular E, D, T, L, S, P or V, particularly preferably E, which have at least the above property a) have.

 (2) For example, the simple mutants R64X-I in which X-1 for E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I, Y, H or N, in particular E, D, T, L, S, P or of V, particularly preferably E, which have at least the above property b).

 (3) For example, the simple mutants R64X-I in which X-1 for E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I, Y, H or N, in particular E, D, T, L, S, P or the V, more preferably E, which have at least the above property c).

(4) For example, the simple mutants R64X-I in which X-1 is E, D, T,

L, S, P, V, K, C, A, G, Q, F, W, I, Y, H or N, in particular E, D, T, L, S, P or the V, more preferably E which have at least the above property d)

 (5) For example, the simple mutants R64X-I in which X-1 for E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I, Y, H or N, in particular E, D, T, L, S, P or of V, particularly preferably E, which have at least the above properties a) and b).

 (6) For example, the simple mutants R64X-I in which X-1 for E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I, Y, H or N, in particular E, D, T, L, S, P or of V, particularly preferably E, which have at least the above properties a) and c).

 (7) For example, the simple mutants R64X-I in which X-1 is E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I, Y, H or N, in particular E, D, T, L, S, P or of V, particularly preferably E, which have at least the above properties a) and d).

 (8) For example, the simple mutants R64X-I in which X-1 is E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I, Y, H or N, in particular E, D, T, L, S, P or of V, particularly preferably E, which have at least the above properties b) and c).

(9) For example, the simple mutants R64X-I in which X-1 is E, D, T,

L, S, P, V, K, C, A, G, Q, F, W, I, Y, H or N, in particular E, D, T, L, S, P or the V, more preferably E which have at least the above properties b) and d).

(10) For example, the simple mutants R64X-I in which X-1 for E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I, Y, H or N, in particular E, D, T, L, S, P or of V, particularly preferably E, which have at least the above properties a) to d). (1 1) For example, mention may be made of the single mutants T17X ₂ in which X _{2 is} an amino acid residue other than T, in particular F, A, I or S, which possess at least the above property a).

(12) Examples which may be mentioned are the simple mutants T17X ₂ in which X _{2 is} an amino acid residue other than T, in particular F, A, I or S, which have at least the above property b).

(13) Examples which may be mentioned are the simple mutants T17X ₂ in which X _{2 is} an amino acid residue other than T, in particular F, A, I or S, which possess at least the above property c).

(14) For example, mention may be made of the single mutants T17X ₂ in which X _{2 is} an amino acid residue other than T, in particular F, A, I, or S, which possess at least the above property d).

(15) Examples which may be mentioned are the simple mutants T17X ₂ in which X _{2 is} an amino acid residue other than T, in particular F, A, I or S, which have at least the above properties a) and b).

(16) Examples which may be mentioned are the simple mutants T17X ₂ in which X _{2 is} an amino acid residue other than T, in particular F, A, I or S, which have at least the above properties a) and c).

(17) For example, the simple mutants T17X ₂ in which X _{2 is} an amino acid residue other than T, in particular F, A, I, or S, which possess at least the above properties a) and d).

(18) Examples which may be mentioned are the simple mutants T17X ₂ in which X _{2 is} an amino acid residue other than T, in particular F, A, I or S, which have at least the above properties b) and c).

(19) Examples which may be mentioned are the simple mutants T17X ₂ in which X _{2 is} an amino acid residue other than T, in particular F, A, I or S, which possess at least the above properties b) and d).

(20) Examples which may be mentioned are the simple mutants T17X ₂ in which X _{2 is} an amino acid residue other than T, in particular F, A, I or S, which have at least the above properties a) to d).

(21) For example, the double mutants R64X- | / G39X ₃ in which X-1 is E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I, Y, H or N, in particular E, D, T, L, S, P or V, preferably E, and X _{3 is} S, A, V, I, L, C, K, Y, F or R, preferred S or A, which possess at least the above property a).

(22) For example, the double mutants R64X- | / G39X ₃ where X-1 is for

E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I, Y, H or N, in particular E, D, T, L, S, P or V. , preferably E, and X _{3 is} S, A, V, I, L, C, K, Y, F or R, preferably Z is S or A, which possess at least the above property b).

(23) For example, the double mutants R64X- | / G39X ₃ in which X-1 is E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I, Y, H or N, in particular E, D, T, L, S, P or V, preferably E, and X ₃ for S, A, V, I, L, C, K, Y, F or R, before - Sgt S or A, stands, which possess at least the above property c).

(24) For example, the double mutants R64X ₁ / G39X ₃ in which X- _{1 is} E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I, Y , H or N, in particular E, D, T, L, S, P or V, preferably E, and X _{3 is} S, A, V, I, L, C, K, Y, F or R, preferably S or A, which possess at least the above property d). (25) For example, the double mutants R64X- | / G39X ₃ where X-ι is for

E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I, Y, H or N, in particular E, D, T, L, S, P or V. , E preferably, and X _{3 is} S, A, V, I, L, C, K, Y, F or R, preferably S or A, which have at least the above properties a) and b).

(26) For example, the double mutants R64X- | / G39X ₃ in which X-1 is E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I, Y, H or N, in particular E, D, T, L,

S, P or V, preferably E, and X _{3 is} S, A, V, I, L, C, K, Y, F or R, preferably S or A, which have at least the above properties a) and c) have.

(27) For example, the double mutants R64X- | / G39X ₃ in which X-1 is E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I, Y, H or N, in particular E, D, T, L, S, P or V, preferably E, and X _{3 is} S, A, V, I, L, C, K, Y, F or R, preferred S or A, which have at least the above properties a) and d).

(28) For example, the double mutants R64X- | / G39X ₃ in which X-1 is E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I, Y, H or N, in particular E, D, T, L, S, P or V, preferably E, and X ₃ for S, A, V, I, L, C, K, Y, F or R, before - Sgt S or A, which have at least the above properties b) and c).

(29) For example, the double mutants R64X- | / G39X ₃ in which X-1 is E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I, Y, H or N, in particular E, D, T, L, S, P or V, preferably E, and X _{3 is} S, A, V, I, L, C, K, Y, F or R, preferred S or A, which have at least the above properties b) and d). (30) For example, the double mutants R64X- | / G39X ₃ where X-ι is for

E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I, Y, H or N, in particular E, D, T, L, S, P or V. , E preferably, and X _{3 is} S, A, V, I, L, C, K, Y, F or R, preferably S or A, which have at least the above properties a) to d).

The exemplary embodiments (1) to (30) listed above relate in particular to mutants of the 7ß-HSDH with SEQ ID NO: 1 1 (optionally N-terminally extended) and have an identity degree of at least 80% or at least 85%, in particular at least 90% up.

2.2 3a-HSDHs: Particular mention should be made of the enzymes described in WO2016 / 023933:

 2.2.1 A 3a-hydroxysteroid dehydrogenase (3a-HSDH) which comprises at least the stereospecific enzymatic reduction of a 3-ketosteroid to the corresponding 3-hydroxysteroid (such as 3,12-diketo-7ß-CS or DHCS to 12-keto-UDCS or 7.12-diketo-3a-CS (7,12-diketo-LCS), in particular with stoichiometric consumption of NADH and / or NADPH, in particular NADH), wherein the enzyme has a mutation in position 34 and / or 68 of SEQ ID NO: 8 or in the corresponding sequence positions of an amino acid sequence derived therefrom having at least 80% sequence identity, such as e.g. At least e.g. 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.5% sequence identity, to SEQ ID NO: 8; and / or C-terminal a chain extension of up to 25, especially up to 15, 14, 13, 12, 11, or 10 identical or different (proteinogenic) amino acid residues, in particular a peptide chain comprising 1 to 10, e.g. 3 to 9 basic amino acid residues, such as lysine, arginine or, in particular, histidine (such as 3xHis, 6xHis or 9xHis); wherein the histidine residues can be attached directly to the C-terminus or via a linker group comprising 1 to 10, in particular 1 to 3, arbitrary amino acid residues. Examples include: LEHHH (SEQ ID NO: 15), LEHHHHHH (SEQ ID NO: 16) and LEHHHHHHHH (SEQ ID NO: 17). Furthermore, the subject matter is those 3a-HSDHs which, in addition to one or more of the above mutations or, alternatively, have an N-terminal chain extension of up to 25 identical or different amino acid residues. These are up to 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 identical or different (proteinogenic) amino acid residues, especially a peptide chain comprising 1 to 10, such as 3 to 9 basic amino acid residues, such as lysine, arginine or, in particular, histidine (such as 3xHis, 6xHis or 9xHis), extended; wherein the histidine residues may be attached directly to the N-terminus or via a 1-10 arbitrary amino acid residue-containing linker group. By way of example, MGSSHHHHHHSSGLVPRGSH- (SEQ ID NO: 18).

 For example, preferred 3a-HSDHs have a mutation in position 34 and / or 68 and are optionally additionally extended N-terminally or C-terminally; or they are only N-terminal or C-terminal extended; each as defined further herein.

 Furthermore, the invention includes native, non-mutated enzyme according to SEQ ID NO: 8, which is modified only N- or C-terminal as described above.

2.2.2 A 3a-HSDH which is at least the stereospecific enzymatic reduction of a Catalyzes 3-ketosteroids to the corresponding 3-hydroxysteroid, and

an amino acid sequence altered by amino acid mutation in SEQ ID NO: 8, wherein the amino acid sequence mutation is selected from single or multiple mutations comprising:

ii. L68X ₂

 wherein Xi is a different, in particular proteinogenic, amino acid residue other than arginine (R), in particular any, especially natural amino acid which increases specific activity and / or reduces substrate inhibition and / or modifies cofactor dependence or cofactor dependency;

 stands; and

X _{2 is} a proteinogenic amino acid residue other than leucine (L), in particular any, especially natural amino acid which increases specific activity and / or reduces substrate inhibition and / or modifies the utilization of cofactor or cofactor; iii. C-terminal to a chain comprising 1 to 10 histidine (H) residues extended addition mutants; wherein, for example, the histidine residues can be attached directly to the C-terminus or via a linker group comprising 1 to 10, in particular 1 to 3, amino acid residues.

 iv. N-terminal chain extended by 1 to 10 histidine (H) residues extended addition mutants; whereby e.g. the histidine residues may be attached directly to the C-terminus or to a linker group comprising 1 to 10 amino acid residues;

 where the mutated, i. altered amino acid sequence has a sequence identity to SEQ ID NO: 8 of 80% to less than 100%, e.g. 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.5% sequence identity.

2.2.3 3a-HSDHs selected under

ca) the simple mutant

 cb) the double mutant

R34X ₁ / L68X ₂

 wherein

 Xi is N, C, S or L;

X _{2 is} A, G, C, K, S or V; particularly preferred combinations of X1, X2; include = N and / or X ₂ = A cc) C-terminally extended addition mutants around a 1 to 10 histidine residues comprising peptide chain; wherein, for example, the histidine residues may be attached directly to the C-terminus or via a 1 to 10, in particular to 3 amino acid residues, linker group;

 cd) N-terminally extended by a 1 to 10 histidine residues comprising peptide chain extended addition mutants; whereby e.g. the histidine residues may be attached directly to the N-terminus or via a 1-10 amino acid residue linker group, and

 ce) mutants according to ca) and cb), in each case additionally containing the addition mutation according to cc) and / or cd), in particular according to cc) or cd). Nonlimiting examples of suitable mutants are

 Comprising single mutants: [R34N], [R34C], [R34S], [R34L], [L68A], [I68C], [L68K], [L68S], [L68V] and [L68G]

 Double mutants comprising: [R34N / L68A]

each without His-tag as well as with C-terminal or N-terminal His-tag.

2.2.4 3a-HSDH as described above under 2.2.1 to 2.2.3, which shows the following property profile in comparison to the native, non-mutated 3a-HSDH with SEQ ID NO: 8:

 a) an increased specific activity (Vmax [U / mg]) for dehydrocholic acid (DHCA) in the enzymatic reduction of DHCA with NADH as cofactor; whereby e.g. the specific activity (U / mg) in the presence of the cofactor NAD (P) H in comparison to the non-mutated enzyme by at least 1, 5, 10, 50 or 100%, but in particular at least 1-fold, especially around 2 to Is increased 100 times or 3 to 20 times or 5 to 10 times

 b) decreased substrate inhibition by DHCA, e.g. with Ki values in the range of> 10mM, e.g. at 1 to 200 mM, 12 to 150 mM, 15 to 100 mM,

 these properties a) and b) may be present individually or in any combination, i. only a), only b), or a) and b).

2.2.5 A 3a-HSDH which comprises at least the stereospecific enzymatic reduction of a 3-ketosteroid to the corresponding 3-hydroxysteroid (such as, for example, 3,12-diketo-7β-CS or DHCS to 12-keto-UDCS or 7,12- Diketo-3a-CS (7,12-diketo-LCS), in particular catalyzed under stoichiometric consumption of NADH and / or NADPH, especially with stoichiometric consumption of NADH, wherein the enzyme has a mutation at position 188 and / or position 185 and optionally position 68 of SEQ ID NO: 8 or in the corresponding Sequence positions of an amino acid sequence with at least 80%, such as. At least such as 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99, 5%, sequence identity to SEQ ID NO: 8; and / or C-terminal a chain extension of up to 15, 14, 13, 12, 11 or 10 identical or different (proteinogenic) amino acid residues, in particular a peptide chain comprising 1 to 10, such as 3 to 9 basic amino acid residues, such as Lysine, arginine or, in particular, histidine (such as 3xHis, 6xHis or 9xHis); wherein the histidine residues may be attached directly to the C-terminus or via a 1 to 3 arbitrary amino acid residue linker group. Examples are: LEHHH, LEHHHHHH and LEHHHHHHHH. Furthermore, the subject matter is those 3a-HSDHs which, in addition to one or more of the above mutations or, alternatively, have an N-terminal chain extension of up to 25 identical or different amino acid residues. These are up to 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 identical or different (proteinogenic) amino acid residues, especially a peptide chain comprising 1 to 10, such as 3 to 9 basic amino acid residues, such as lysine, arginine or especially histidine (such as 3xHis, 6xHis or 9xHis) extended; wherein the histidine residues can be attached directly to the N-terminus or via a 1 to 10 arbitrary amino acid residues comprising linker group. By way of example: MGSSHHHHHHSSGLVPRGSH-.

 For example, preferred 3a-HSDHs each have a mutation at position 188 and / or 185 and optionally at position 68 and are optionally additionally extended N-terminally or C-terminally; or they are only N-terminal or C-terminal extended; each as defined further herein.

2.2.6 3a-HSDH which is at least the stereospecific enzymatic reduction of a

Ketosteroids catalyzed to the corresponding 3-hydroxysteroid, and

an amino acid sequence altered by amino acid mutation in SEQ ID NO: 8, wherein the amino acid sequence mutation is selected from single or multiple mutations comprising:

b) T188X ₃

and optionally in addition

where Xi is a proteinaceous amino acid residue other than proline (P); X _{2 is} a (proteinogenic) amino acid residue other than leucine (L); X _{3 is} a proteinaceous amino acid residue other than threonine (T); d) optionally additionally with C-terminal extension comprising 1 to

10 histidine (H) residues; and / or optionally additionally with N-terminal extension comprising 1 to 10 histidine (H) radicals.

where the mutated, i. altered amino acid sequence has a sequence identity to SEQ ID NO: 8 of 80% to less than 100%, e.g. 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.5% sequence identity. 2.2.7 3a-HSDH as described above under 2.2.5 and 2.2.6, selected at

 a) the simple mutants

T188X ₃

b) the double mutants

 wherein

 Xi is A, T, S, G or V, especially A;

X _{2 is} A, G, C, K, S or V, especially A; and

X _{3 is} A, G, W, S or V, especially A or S;

 c) optionally additionally with C-terminal extension comprising 1 to 10 histidine residues, where e.g. the histidine residues may be attached directly to the C-terminus or via a 1-10 amino acid residue linker group.

 and; and or

 d) optionally additionally with N-terminal extension comprising 1 to 10 histidine residues; whereby e.g. the histidine residues may be attached directly to the N-terminus or via a 1-10 amino acid residue linker group.

 and

 e) mutants according to a) and b), each additionally containing the addition mutation according to c) and / or d), in particular according to c) or d).

Particularly noteworthy are the double mutants P185Xi / T188X ₃ ; L68X ₂ / T188X ₃ ; L68X ₂ / T185Xi; without or with N- or C-terminal extension as defined above.

Nonlimiting examples of suitable mutants are:

Comprising single mutants: [T188A], [T188S], [T188G], [T188V], [T188W], [P185A], [P185T], [P185S], [P185G], [P185V], [P185A],

 Double mutants comprising: [L68A / T188A] and [L68A / T188S]

each without His-tag as well as with C-terminal or N-terminal His-tag.

2.2.8 3a -HSDH as described above under 2.2.5 to 2..2.7, which shows the following property profile in comparison to the native, non-mutated 3a-HSDH with SEQ ID NO: 8:

a) an increased specific activity (v _max [U / mg]) for dehydrocholic acid (DHCA) in the enzymatic reduction of DHCA with NADH as cofactor; where, for example, the specific activity (U / mg) in the presence of the cofactor NADH in comparison to the non-mutated enzyme is at least 1, 5, 10, 50 or 100%, but in particular at least 1-fold, especially around 2 to 100 is increased by a factor of 3 to 20 or 5 to 10 times,

b. decreased substrate inhibition by DHCA, such as with Ki values in the range of> 10 mM, such as 11 to 200 mM, 12 to 150 mM, 15 to 100 mM, c. an increased specific activity (v _max [U / mg]) for NADH in the enzymatic reduction of DHCA with NADH as cofactor. where, for example, the specific activity (U / mg) in the presence of the DHCA compared to the non-mutated enzyme by at least 1, 5, 10, 50 or 100%, but in particular at least by 1-fold, in particular by 2 to 100 times or 3 to 20 times or 5 to 10 times,

 these properties a) to c) may be present individually or in any combination, i. only a), only b), only c), a) and b), a) and c) or b) and c).

3. Nucleic acids and constructs

3. 1 nucleic acids

 The invention also nucleic acid sequences encoding an enzyme with 7ß-HSDH, ADH, FDH, GDH and / or 3a-HSDH activity and their mutants.

 The present invention also relates to nucleic acids having a certain degree of identity to the specific sequences described herein.

 "Identity" between two nucleic acids is understood to mean the identity of the nucleotides over the entire nucleic acid length, in particular the identity which is determined by comparison with the Vector NTI Suite 7.1 software from Informax (USA) using the Clustal method (Higgins DG, Sharp Fast and sensitive multiple sequence alignments on a microcomputer, Comput Appl. Biosci 1989 Apr; 5 (2): 151 -1) is calculated with the following parameters:

Multiple alignment parameters: Gap opening penalty 10

 Gap extension penalty 10

 Gap Separation penalty ranks 8

 Gap separation penalty off

% identity for alignment delay 40

 Residue specific gaps off

 Hydrophilic residues gap off

 Transition weighing 0 Pairwise alignment parameter:

 FAST algorithm on

 K-tuplesize 1

 Gap penalty 3

 Window size 5

Number of best diagonals 5

Alternatively, the identity may also be after Chenna, Ramu, Sugawara, Hideaki, Koike, Tadashi, Lopez, Rodrigo, Gibson, Toby J, Higgins, Desmond G, Thompson, Julie D. Multiple sequence alignment with the Clustal series of programs. (2003) Nucleic Acids Res 31 (13): 3497-500, according to Internet address: http://www.ebi.ac.uk/Tools/clustalw/index.html# and determined with the following parameters:

DNA Gap Open Penalty 15.0

 DNA Gap Extension Penalty 6.66

 DNA Matrix Identity

 Protein Gap Open Penalty 10.0

 Protein Gap Extension Penalty 0.2

 Protein matrix benefits

 Protein / DNA ENDGAP -1

 Protein / DNA GAPDIST 4

All nucleic acid sequences mentioned herein (single- and double-stranded DNA and RNA sequences, such as cDNA and mRNA) can be prepared in a manner known per se by chemical synthesis from the nucleotide units, for example by fragment condensation of individual overlapping, complementary nucleic acid units of the double helix. The chemical synthesis of oligonucleotides can be carried out, for example, in a known manner by the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, Pages 896-897). The attachment of synthetic oligonucleotides and filling gaps with the aid of the Klenow fragment of the DNA polymerase and ligation reactions and general cloning methods are described in Sambrook et al. (1989), Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press.

 The invention also relates to nucleic acid sequences (single- and double-stranded DNA and RNA sequences, such as cDNA and mRNA) coding for one of the above polypeptides and their functional equivalents, which are e.g. accessible using artificial nucleotide analogs.

 The invention relates both to isolated nucleic acid molecules which code for polypeptides or proteins or biologically active portions thereof according to the invention, as well as nucleic acid fragments which are e.g. for use as hybridization probes or primers for the identification or amplification of coding nucleic acids of the invention.

 The nucleic acid molecules according to the invention can additionally contain untranslated sequences from the 3 'and / or 5' end of the coding gene region

 The invention further comprises the nucleic acid molecules complementary to the specifically described nucleotide sequences or a portion thereof.

 The nucleotide sequences of the invention enable the generation of probes and primers useful for the identification and / or cloning of homologous sequences in other cell types and organisms. Such probes or primers usually comprise a nucleotide sequence region which is under "stringent" conditions (see below) at least about 12, preferably at least about 25, such as about 40, 50 or 75 consecutive nucleotides of a sense strand of a nucleic acid sequence of the invention or a corresponding nucleic acid sequence Antisense strands hybridizes.

 An "isolated" nucleic acid molecule is separated from other nucleic acid molecules present in the natural source of the nucleic acid and, moreover, may be substantially free of other cellular material or culture medium when produced by recombinant techniques, or free from chemical precursors or other chemicals if it is synthesized chemically.

 A nucleic acid molecule according to the invention can be analyzed by means of molecular biological

Standard techniques and the sequence information provided according to the invention are isolated. For example, cDNA can be isolated from a suitable cDNA library by using one of the specifically disclosed complete sequences, or a portion thereof, as a hybridization probe and standard hybridization techniques (as described, for example, in Sambrook, J., Fritsch, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Moreover, a nucleic acid molecule comprising one of the disclosed sequences or a portion thereof, isolate by polymerase chain reaction, wherein the oligonucleotide primers that were created on the basis of this sequence can be used. The thus amplified nucleic acid can be cloned into a suitable vector and characterized by DNA sequence analysis. The oligonucleotides according to the invention can furthermore be prepared by standard synthesis methods, for example using an automatic DNA synthesizer.

 Nucleic acid sequences according to the invention or derivatives thereof, homologs or parts of these sequences, can be prepared, for example, by conventional hybridization methods or the PCR technique from other bacteria, e.g. isolate via genomic or cDNA libraries. These DNA sequences hybridize under standard conditions with the sequences according to the invention.

 By "hybridizing" is meant the ability of a poly- or oligonucleotide to bind to a nearly complementary sequence under standard conditions, while under these conditions non-specific binding between non-complementary partners is avoided. For this, the sequences may be 90-100% complementary. The property of complementary sequences to be able to specifically bind to one another, for example, in the Northern or Southern Blot technique or in the primer binding in PCR or RT-PCR advantage.

 For hybridization, it is advantageous to use short oligonucleotides of the conserved regions. However, it is also possible to use longer fragments of the nucleic acids according to the invention or the complete sequences for the hybridization. Depending on the nucleic acid used (oligonucleotide, longer fragment or complete sequence) or which nucleic acid type DNA or RNA is used for the hybridization, these standard conditions vary. For example, the melting temperatures for DNA: DNA hybrids are about 10 ° C lower than those of DNA: RNA hybrids of the same length.

Under standard conditions, for example, depending on the nucleic acid temperatures between 42 and 58 ° C in an aqueous buffer solution with a concentration between 0.1 to 5 x SSC (1 x SSC = 0.15 M NaCl, 15 mM sodium, pH 7.2) or additionally in the presence of 50% formamide such as 42 ° C in 5 x SSC, 50% formamide to understand. Advantageously, the hybridization conditions for DNA: DNA hybrids are 0.1X SSC and temperatures between about 20 ° C to 45 ° C, preferably between about 30 ° C to 45 ° C. For DNA: RNA hybrids, the hybridization conditions are advantageously 0.1 x SSC and temperatures between about 30 ° C to 55 ° C, preferably between about 45 ° C to 55 ° C. These indicated temperatures for the hybridization are exemplary calculated melting temperature values for a nucleic acid with a length of about 100 nucleotides and a G + C content of 50% in the absence of formamide. The experimental conditions for DNA hybridization are described in relevant textbooks of genetics, such as, for example, Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989, and formulas which are known to the person skilled in the art are dependent on the length of the nucleic acids, for example. calculate the type of hybrid or G + C content. Further information on hybridization can be found in the following textbooks by the person skilled in the art: Ausubel et al. (eds), 1985, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Harnes and Higgins (eds), 1985, Nucleic Acids Hybridization: A Practical Approach, IRL Press at Oxford University Press, Oxford; Brown (ed), 1991, Essential Molecular Biology: A Practical Approach, IRL Press at Oxford University Press, Oxford.

 The "hybridization" can in particular take place under stringent conditions Such hybridization conditions are described, for example, in Sambrook, J., Fritsch, EF, Maniatis, T., in: Molecular Cloning (A Laboratory Manual), 2nd edition, Cold Spring Harbor Laboratory Press, 1989, pp. 9.31-9.57 or in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6.

 By "stringent" hybridization conditions are meant in particular: The incubation at 42 ° C overnight in a solution consisting of 50% formamide, 5 x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate ( pH7.6), 5x Denhardt's solution, 10% dextran sulfate and 20 g / ml denatured salmon sperm DNA, followed by a filter wash with 0.1 x SSC at 65 ° C.

 The invention also relates to derivatives of the specifically disclosed or derivable nucleic acid sequences.

 Thus, further nucleic acid sequences of the invention may be used e.g. are derived from SEQ ID NO: 7, 10 or 13 and differ therefrom by addition, substitution, insertion or deletion of single or multiple nucleotides, but furthermore encode polypeptides having the desired property profile.

 Also included according to the invention are those nucleic acid sequences which comprise so-called silent mutations or which have changed in accordance with the codon usage of a specific source or host organism, in comparison with a specifically mentioned sequence, as well as naturally occurring variants, such as e.g. Splice variants or allelic variants, of which

 Articles are also provided by conservative nucleotide substitutions (i.e., the amino acid in question is replaced by an amino acid of like charge, size, polarity, and / or solubility).

The invention also relates to the molecules derived by sequence polymorphisms from the specifically disclosed nucleic acids. These genetic polymorphisms can occur between individuals within a population due to the natural varia- exist. These natural variations usually cause a variance of 1 to 5% in the nucleotide sequence of a gene.

 Derivatives of the nucleic acid sequence according to the invention with the sequence SEQ ID NO: 7, 10 or 13 are, for example, allelic variants which have at least 60% homology at the derived amino acid level, preferably at least 80% homology, most preferably at least 90% homology over the entire sequence range (for homology at the amino acid level, see the above comments on the polypeptides). About partial regions of the sequences, the homologies may be advantageous higher.

 Furthermore, derivatives are also to be understood as meaning homologs of the nucleic acid sequences according to the invention, in particular of SEQ ID NO: 7, 10 or 13, for example fungal or bacterial homologs, truncated sequences, single stranded DNA or RNA of the coding and noncoding DNA sequence. Thus, e.g. Homologues to the SEQ ID NO: 7, 10 or 13 at the DNA level a homology of at least 40%, preferably of at least 60%, more preferably of at least 70%, most preferably of at least 80% over the entire in SEQ ID NO : 7, 10 or 13 indicated DNA range.

 In addition, by derivatives, for example, to understand fusions with promoters. The promoters, which are upstream of the specified nucleotide sequences, can be modified by at least one nucleotide exchange, at least one insertions, inversions and / or deletions, without, however, impairing the functionality or effectiveness of the promoters. In addition, the promoters can be increased by changing their sequence in their effectiveness or completely replaced by more effective promoters of alien organisms.

 In addition, methods for generating functional mutants are known to the person skilled in the art.

 Depending on the technique used, the person skilled in the art can introduce completely random or even more targeted mutations into genes or non-coding nucleic acid regions (which are important, for example, for the regulation of expression) and then generate gene banks. The molecular biological methods required for this purpose are known to the person skilled in the art and are described, for example, in Sambrook and Russell, Molecular Cloning. 3rd Edition, Cold Spring Harbor Laboratory Press 2001.

 Methods for the modification of genes and thus for the modification of the protein coded by these have long been familiar to the person skilled in the art, for example

the site-specific mutagenesis, in which one or more nucleotides of a gene are exchanged in a targeted manner (Trower MK (ed.) 1996, in vitro mutagenesis protocols, Humana Press, New Jersey), the saturation mutagenesis, in which a codon for any amino acid can be exchanged or added at any position of a gene (Kegler-Ebo DM, Docktor CM, DiMaio D (1994) Nucleic Acids Res 22: 1593, Barettino D, Feigenbutz M, Valcärel R, Stunnenberg HG (1994) Nucleic Acids Res 22: 541, Barik S (1995) Mol Biotechnol 3: 1),

 the error-prone polymerase chain reaction (error-prone PCR), in which nucleotide sequences are mutated by defective DNA polymerases (Eckert KA, Kunkel TA (1990) Nucleic Acids Res 18: 3739);

 - The passage of genes in mutator strains in which, for example, due to deficient DNA repair mechanisms, an increased mutation rate of nucleotide sequences occurs

(Greener A, Callahan M, Jerpseth B (1996) An efficient random mutagenesis technique using E. coli mutator strain In: Trower MK (ed.) In Vitro Mutagenesis Protocols, Humana Press, New Jersey), or

 DNA shuffling, in which a pool of closely related genes is formed and digested, and the fragments are used as templates for a polymerase chain reaction in which repeated strand separation and recapture ultimately generate full-length mosaic genes (Stemmer WPC (1994) Nature 370: 389; Stemmer WPC (1994) Proc Natl Acad. USA 91: 10747).

 Using directed evolution (described, inter alia, in Reetz MT and Jaeger KE (1999), Topics Curr Chem 200: 31, Zhao H, Moore JC, Volkov AA, Arnold FH (1999), Methods In: Demain AL, Davies JE (eds.) Manual of industrial microbiology and biotechnology, the expert can also generate functional mutants in a targeted manner and also on a large scale In a first step, first gene libraries of the respective proteins are generated, using, for example, the abovementioned methods The gene banks are appropriately expressed, for example by bacteria or by phage display systems.

The respective genes of host organisms expressing functional mutants with intrinsic properties that largely correspond to the desired properties can be subjected to another round of mutation. The steps of mutation and selection or screening can be repeated iteratively until the functional mutants present have the desired properties to a sufficient extent. By means of this iterative procedure, a limited number of mutations, such as 1 to 5 mutations, can be carried out step by step and evaluated and selected for their influence on the relevant enzyme property. The selected mutant can then be subjected in the same way to a further mutation step. By doing so leaves significantly reduce the number of single mutants to be studied.

 The results according to the invention provide important information regarding the structure and sequence of the enzymes in question, which are required in order to selectively generate further enzymes with desired modified properties. In particular, so-called "hot spots" can be defined, that is to say sequence sections which are potentially suitable for modifying an enzyme property by introducing targeted mutations.

3.2 Constructs

 The invention also relates to expression constructs comprising, under the genetic control of regulatory nucleic acid sequences, a nucleic acid sequence coding for at least one polypeptide according to the invention; and vectors comprising at least one of these expression constructs.

 According to the invention, an "expression unit" is understood as meaning a nucleic acid with expression activity which comprises a promoter as defined herein and, after functional linkage with a nucleic acid or a gene to be expressed, regulating the expression, ie the transcription and the translation, of this nucleic acid or gene In this context, therefore, one speaks of a "regulatory nucleic acid sequence". In addition to the promoter, other regulatory elements, e.g. Enhancers, be included.

 An "expression cassette" or "expression construct" is understood according to the invention to mean an expression unit which is functionally linked to the nucleic acid to be expressed or to the gene to be expressed. Thus, unlike an expression unit, an expression cassette comprises not only nucleic acid sequences that regulate transcription and translation, but also the nucleic acid sequences that are to be expressed as a protein as a result of transcription and translation.

 The terms "expression" or "overexpression" in the context of the invention describe the production or increase of the intracellular activity of one or more enzymes in a microorganism which are encoded by the corresponding DNA, for example by introducing a gene into an organism replace existing gene with another gene, increase the copy number of the gene (s), use a strong promoter or use a gene coding for a corresponding enzyme with a high activity, and optionally combine these measures.

 Such constructs according to the invention preferably comprise a promoter 5'-upstream of the respective coding sequence and a terminator sequence 3'-downstream and optionally further customary regulatory elements, in each case operatively linked to the coding sequence.

Under a "promoter", a "nucleic acid with promoter activity" or a "Pro According to the invention, "motor sequence" is understood as meaning a nucleic acid which, in functional linkage with a nucleic acid to be transcribed, regulates the transcription of this nucleic acid.

 A "functional" or "operative" linkage in this context means, for example, the sequential arrangement of one of the nucleic acids with promoter activity and a nucleic acid sequence to be transcribed and, if appropriate, further regulatory elements, for example nucleic acid sequences which ensure the transcription of nucleic acids, and, for example a terminator such that each of the regulatory elements can fulfill its function in the transcription of the nucleic acid sequence. This does not necessarily require a direct link in the chemical sense. Genetic control sequences, such as enhancer sequences, may also exert their function on the target sequence from more distant locations or even from other DNA molecules. Preferred are arrangements in which the nucleic acid sequence to be transcribed is positioned behind (i.e., at the 3 'end) of the promoter sequence such that both sequences are covalently linked together. The distance between the promoter sequence and the transgenic nucleic acid sequence to be expressed may be less than 200 base pairs, or less than 100 base pairs or less than 50 base pairs.

 In addition to promoters and terminators, examples of other regulatory elements include targeting sequences, enhancers, polyadenylation signals, selectable markers, amplification signals, origins of replication, and the like. Suitable regulatory sequences are e.g. in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).

 Nucleic acid constructs according to the invention comprise in particular sequence SEQ ID NO: 7, 10 or 13 or derivatives and homologs thereof, as well as the nucleic acid sequences derivable therefrom, which are advantageously used with one or more regulatory signals for the control, e.g. Increased, the gene expression was operatively or functionally linked.

In addition to these regulatory sequences, the natural regulation of these sequences may still be present before the actual structural genes and may have been genetically altered so that natural regulation is eliminated and expression of genes increased. However, the nucleic acid construct can also have a simpler structure, ie no additional regulatory signals were inserted before the coding sequence and the natural promoter with its regulation was not removed. Instead, the natural regulatory sequence is mutated so that regulation stops and gene expression is increased. A preferred nucleic acid construct advantageously also contains one or more of the already mentioned "enhancer" sequences, functionally linked to the promoter, which allow increased expression of the nucleic acid sequence. Additional advantageous sequences can also be inserted at the 3 'end of the DNA sequences, such as further regulatory elements or terminators. The nucleic acids of the invention may be contained in one or more copies in the construct. The construct may also contain further markers, such as antibiotic resistance or auxotrophic complementing genes, optionally for selection on the construct.

Examples of suitable regulatory sequences are promoters such as cos-, tac-, trp-, tet-, trp-tet, Ipp-, lac-, Ipp-lac-, laclq ^" T7-, T5-, T3-, gal-, trc, ara, rhaP (rhaP _B AD) SP6, lambda P _R - or contained in the lambda P _L promoter, which are advantageously used in gram-negative bacteria, Further advantageous regulatory sequences are, for example, in the gram-positive Promoters amy and SP02, in the yeast or fungal promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH.It is also possible to use artificial promoters for regulation.

 The nucleic acid construct, for expression in a host organism, is advantageously inserted into a vector, such as a plasmid or a phage, which allows for optimal expression of the genes in the host. Vectors other than plasmids and phages are also all other vectors known to those skilled in the art, e.g. Viruses such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosides, and linear or circular DNA. These vectors can be autonomously replicated in the host organism or replicated chromosomally. These vectors represent a further embodiment of the invention.

Suitable plasmids are described, for example, in E. coli pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, plN-III ¹¹³ -B1, Agt1 1 or pBdCI, in Streptomyces plJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB1 10, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667, in fungi pALS1, pIL1 or pBB1 16, in yeasts 2alphaM, pAG-1, YEp6, YEp13 or pEMBLYe23 or in plants pLGV23, pGHIac ⁺ , pBIN19, pAK2004 or pDH51. The plasmids mentioned represent a small selection of the possible plasmids. Further plasmids are well known to the person skilled in the art and can be found, for example, in the book Cloning Vectors (Eds. Pouwels PH et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).

In a further embodiment of the vector, the vector containing the nucleic acid construct according to the invention or the nucleic acid according to the invention can also advantageously be introduced into the microorganisms in the form of a linear DNA and integrated into the genome of the host organism via heterologous or homologous recombination. be grated. This linear DNA can consist of a linearized vector such as a plasmid or only of the nucleic acid construct or of the nucleic acid according to the invention.

 For optimal expression of heterologous genes in organisms, it is advantageous to modify the nucleic acid sequences according to the specific "codon usage" used in the organism. The "codon usage" can be easily determined by computer evaluations of other known genes of the organism concerned.

 An expression cassette according to the invention is produced by fusion of a suitable promoter with a suitable coding nucleotide sequence and a terminator or polyadenylation signal. For this purpose, common recombination and cloning techniques are used, as described, for example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusion, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).

 The recombinant nucleic acid construct or gene construct is advantageously injected into a host-specific vector for expression in a suitable host organism, which enables optimal expression of the genes in the host. Vectors are well known to those skilled in the art and can be found, for example, in "Cloning Vectors" (Pouwels P.H. et al., Eds. Elsevier, Amsterdam-New York-Oxford, 1985).

 In particular:

 Nucleotide sequences encoding a 3a-HSDH or a 7ß-HSDH according to one of the embodiments described above.

By way of example, nucleotide sequences selected from among sequences

a) simultaneously coding for a GDH and a 3a-HSDH as described above and optionally a 7ß-HSDH as described above;

b) coding for a fusion protein comprising a GDH and a 3a -HSDH as described above and optionally a 7β-HSDH as described above;

wherein the coding sequences can be contained independently of one another one or more times in the construct, such as in 2, 3, 4, 5, or 6 to 10 copies. By choosing the appropriate copy number, any differences in activity in the individual expression products can thus be compensated. Expression cassettes comprising, under the control of at least one regulatory sequence, at least one nucleotide sequence as described above and optionally coding sequences for at least one (such as 1, 2 or 3) further enzyme selected from among hydrogen roxysteroid dehydrogenases, in particular 7β-HSDH as described herein, and dehydrogenases suitable for cofactor regeneration, such as FDH, GDH, ADH, G-6-PDH, PDH. In particular, the enzymes contained in an expression cassette can use different, but preferably identical cofactor pairs, such as, for example, the cofactor pair NAD + / NADH or NADP + / NADPH.

4. microorganisms

 Depending on the context, the term "microorganism" may be understood to mean the starting microorganism (wild-type) or a genetically modified, recombinant microorganism or both.

 With the aid of the vectors according to the invention, recombinant microorganisms can be produced, which are transformed, for example, with at least one vector according to the invention and can be used to produce the polypeptides according to the invention. Advantageously, the above-described recombinant constructs according to the invention are introduced into a suitable host system and expressed. In this case, it is preferred to use familiar cloning and transfection methods known to the person skilled in the art, for example co-precipitation, protoplast fusion, electroporation, retroviral transfection and the like, in order to express the stated nucleic acids in the respective expression system. Suitable systems are described, for example, in Current Protocols in Molecular Biology, F. Ausubel et al., Ed., Wiley Interscience, New York 1997, or Sambrook et al. Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. For example, review of bacterial expression systems for heterologous expression of proteins provides. also Terpe, K. Appl. Microbiol. Biotechnol. (2006) 72: 21 1 - 222.

In principle, all prokaryotic or eukaryotic organisms are suitable as recombinant host organisms for the nucleic acid or nucleic acid construct according to the invention. Advantageously, microorganisms such as bacteria, fungi or yeast are used as host organisms. Advantageous are gram-positive or gram-negative bacteria, preferably bacteria of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae or Nocardiaceae, more preferably bacteria of the genera Escherichia, Pseudomonas, Comamonas, Streptomyces, Nocardia, Burkholderia, Salmonella, Agrobacterium, Clostridium or Rhodococcus used. Very particularly preferred is the genus and species Escherichia coli. Further beneficial bacteria are also found in the group of alpha-proteobacteria, beta-proteobacteria or gamma-proteobacteria The host organism or host organisms according to the invention preferably contain at least one of the nucleic acid sequences, nucleic acid constructs or vectors described in this invention, which code for an enzyme with 7β-HSDH activity as defined above.

 The organisms used in the method according to the invention are grown or grown according to the host organism in a manner known to those skilled in the art. Microorganisms are usually in a liquid medium containing a carbon source usually in the form of sugars, a nitrogen source usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as iron, manganese, magnesium salts and optionally vitamins, at temperatures between 0 ° C and 100 ° C, preferably between 10 ° C to 60 ° C attracted under oxygen fumigation. In this case, the pH of the nutrient fluid can be kept at a fixed value, that is regulated during the cultivation or not. The cultivation can take place batchwise, semi-batchwise or continuously. Nutrients can be presented at the beginning of the fermentation or fed in semi-continuously or continuously.

For example:

 Recombinant microorganisms carrying at least one nucleotide sequence or at least one expression cassette or at least one expression vector, each as described above.

 Recombinant microorganisms carrying in addition to the coding sequence for 3a and 7ß-HSDH the coding sequence for a dehydrogenase suitable for cofactor regeneration, wherein the dehydrogenase is selected from NADPH regenerating enzymes, such as NADPH dehydrogenases, alcohol dehydrogenases (ADH), and NADPH re Generating formate dehydrogenases (FDH), as well as glucose dehydrogenase (GDH), glucose co-6-phosphate dehydrogenase (G-6-PDH), or phosphite dehydrogenases (PtDH), or NADH-regenerating enzymes, such as NADH dehydrogenases, NADH regenerating formate dehydrogenases (FDH), NADH regenerating alcohol dehydrogenases (ADH), NADH regenerating glucose-6-phosphate dehydrogenases (G6PDH), NADH regenerating phosphite dehydrogenases (PtDH) and NADH regenerating glucose dehydrogenases (GDH).

Recombinant microorganisms which carry the coding sequences for a 3a-HSDH, a GDH and a 7ß-HSDH (or their mutants) on one or more (different) expression constructs. The invention thus relates to recombinant microorganisms which are modified with a single-plasmid system (such as transformed, for example), which contain the coding sequences for 3a -HSD, GDH and 7ß-HSDH (or their mutants) in one or more copies, such as in 2, 3, 4, 5, or 6 to 10 copies, wear. The invention thus also relates to recombinant microorganisms which are modified (such as transformed) with a single-plasmid system which contains the coding sequences for 7ß-HSDH, GDH and 3a-HSDH (or their mutants) in one or more copies, such as in 2, 3, 4, 5 or 6 to 10 copies. However, the enzymes (7β-HSDH, GDH and 3a-HSDH or their mutants) can also be present on 2 or 3 separate, mutually compatible plasmids in one or more copies. Suitable base vectors for the production of single-plasmid systems and multicopy plasmids are known to the person skilled in the art. Examples which may be mentioned are for a single-plasmid system, for example pET21 a and for multicopy plasmids, for example the Duet vectors marketed by Novagen, such as pACYCDuet-1, pETDuet-1, pCDFDuet-1, pRSFDuet-1 and pCOLADuet-1. Such vectors, their compatibility with other vectors and microbial host strains can be found, for example, in the "User Protocol TB340 Rev. E0305 from Novagen.

 The optimum combination of enzymes for the generation of plasmid systems can be carried out by the person skilled in the art without taking reasonable account of the teaching of the present invention. Thus, for example, depending on the cofactor specificity of the particular HSDH enzyme used, the person skilled in the art can select the enzyme most suitable for cofactor regeneration, selected from the abovementioned dehydrogenases, in particular GDH and the respective mutants thereof.

 Furthermore, it is possible to distribute the enzymes chosen for the reaction to two or more plasmids and to produce two or more different recombinant microorganisms with the plasmids thus prepared, which are then used together for the biocatalytic reaction according to the invention. The particular enzyme combination used for the preparation of the plasmid can be carried out in particular under the specification of a comparable cofactor use. For example, a first microorganism may be modified with a plasmid carrying the coding sequence for a 7β-HSDH or mutant thereof and a GDH. On the other hand, a second microorganism may be modified with a plasmid carrying the coding sequence for a 3α-HSDH or mutant thereof and the coding sequence for a GDH. Both enzyme pairs can be chosen so that they can regenerate the same cofactor pairs. Both microorganisms can then be used simultaneously for the biocatalytic reaction according to the invention.

 The use of two separate biocatalysts (recombinant microorganisms) can offer two major advantages over the use of only one biocatalyst in which all synthetic enzymes are expressed:

a) Both biocatalysts can be genetically modified and optimized separately from each other. In particular, it is possible to use various cofactor regeneration enzymes optimized for either NADH or NADPH regeneration. b) For biocatalysis, the biocatalysts can be used in different proportions. This allows intervention in the individual reaction velocities of the multi-enzyme process during biocatalysis, even after all biocatalysts have already been prepared.

 The recombinant microorganisms described herein can also be used

HSDH knock-out strain, prepared as e.g. described in WO201 1/147957 of the applicant.

5. Production of UDCS

1st step: Chemical oxidation of CDCA to 3, 7-diketo-UDCA

 The reaction is carried out using methods known per se for the chemical oxidation of secondary alcohols. For example, the reaction by means of potassium dichromate in dilute sulfuric acid, by means of pyridinium dichromate, by means of Jones reagent, or Collins reagent (cf., for example, Brückner, Reaction mechanisms, 3rd edition, 2004, Spektrum Akademischer Verlag

5.1 SWERN oxidation

For this purpose, oxalyl chloride is dissolved in CH ₂ Cl ₂ and cooled with an acetone / T rockeneis bath to -78 ° C. To this is added dropwise a solution of dimethyl sulfoxide CH ₂ Cl ₂ added dropwise. A solution of chenodeoxycholic acid (CDCA) in a mixture of CH ₂ Cl ₂ / DMSO is added. The reaction mixture is stirred, then triethylamine is added, stirring is continued and the cooling bath is removed. To the heated to RT reaction mixture is added H ₂ 0 and stirred. Subsequently, aqueous and organic phases are separated and the aqueous phase CH ₂ Cl _{2 is} extracted. The combined organic phases are washed successively with 0.5 M HCl and dist. H ₂ 0, dried over NaS0 ₄ and freed from the solvent in vacuo.

5.2 Tempo oxidation For this purpose, CDCA is dissolved in acetonitrile. After cooling the reaction solution to 4 ° C, sodium bromide and TEMPO are added. Subsequently, a pre-cooled NaOCl solution is added in aliquots with constant stirring to the reaction solution, wherein the internal temperature was kept below 5 ° C. After completion of the reaction, the reaction mixture is brought to room temperature, treated with HCl, which dissolves solid formed and then extracted with ethyl acetate. The combined extracts were with HCl, dried over MgS0 ₄ and freed from the solvent. 3,7-diketo-UDCA was obtained as a colorless solid.

5.3 Oxidation by sodium hypochlorite / sodium bisulfite

The chenodeoxycholic acid is dissolved in acetic acid at 18 ° C. The hypochlorite is added to the resulting brown solution. The temperature is kept at 7 -15 ° C. The milky suspension was then stirred with cooling. To neutralize the excess hypochlorite, sodium bisulfite is added thereto. The temperature is kept constant at 15 ° C and stirred. After addition of H ₂ 0 a milky suspension is obtained, which is heated to 70 ° C and then cooled. The product is filtered off and washed with water. The product is then dried.

2nd step: biocatalytic, i. enzymatic or microbial conversion of 3, 7-diketo-UDCA to UDCA

 In aqueous solution, 3,7-diketo-UDCA is specifically reduced by 3a-HSDH and 7β-HSDH or mutants thereof to UDCS in the presence of NADPH and / or NADH. The cofactor NADPH or NADH can be replaced by an ADH or FDH or GDH or mutants thereof with consumption of e.g. Isopropanol (ADH) or Natiumformiat (FDH) or Gluco- se (GDH) are regenerated. The reaction is under mild condition. For example, the reaction at pH = 6 to 9, in particular about pH = 8 and at about 10 to 30, 15 to 25 or about 23 ° C are performed.

In the case of a microbial reaction step, recombinant microorganisms expressing the required enzyme activity (s) in the presence of the substrate to be reacted (3,7-diketo-UDCA) may be cultured anaerobically or aerobically in suitable liquid media. Suitable cultivation conditions are known per se to the person skilled in the art. They include reactions in the pH range of for example 5 to 10 or 6 to 9, at temperatures in the range of 10 to 60 or 15 to 45 or 25 to 40 or 37 ° C. Suitable media include z, B, LB and TB or the media described below. The reaction can be carried out z, B, batchwise or continuously or in other conventional process variants (as described above). The reaction time can be, for example, in the range of minutes to several hours or days, and for example be 1 h to 48 h. Optionally, when enzyme activity is not continuously expressed, it may be introduced by addition of a suitable inducer upon reaching a target cell density, eg, from about OD ₆ oo = 0.5 to 1.0.

Further possible suitable modifications of the microbial production process with regard to fermentation, additives to the medium, enzyme immobilization and Isolation of valuable substances can also be found in the following section concerning "Recombinant production of the enzymes or mutants"

6. Recombinant production of the enzymes and mutants

 The invention furthermore relates to processes for the recombinant production of polypeptides according to the invention or functional, biologically active fragments thereof, wherein a polypeptide-producing microorganism is cultivated, if appropriate, the expression of the polypeptides is induced and these are isolated from the culture. The polypeptides can thus also be produced on an industrial scale, if desired.

 The microorganisms produced according to the invention can be cultured continuously or batchwise in the batch process (batch cultivation) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process). A summary of known cultivation methods is in the textbook by Chmiel (Bioprozesstechnik 1. Introduction to bioprocess engineering (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (bioreactors and peripheral facilities (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)) Find.

 The culture medium to be used must suitably satisfy the requirements of the respective strains. Descriptions of culture media of various microorganisms are contained in the Manual of Methods for General Bacteriology of the American Society for Bacteriology (Washington D.C, USA, 1981).

 These media which can be used according to the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.

 Preferred carbon sources are sugars, such as mono-, di- or polysaccharides. Examples of very good carbon sources are glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugar can also be added to the media via complex compounds, such as molasses, or other by-products of sugar refining. It may also be advantageous to add mixtures of different carbon sources. Other possible sources of carbon are oils and fats such. B. soybean oil. Sunflower oil. Peanut oil and coconut fat, fatty acids such. As palmitic acid, stearic acid or linoleic acid, alcohols such. As glycerol, methanol or ethanol and organic acids such. As acetic acid or lactic acid.

Nitrogen sources are usually organic or inorganic nitrogen compounds or materials containing these compounds. Exemplary nitrogen sources include ammonia gas or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as corn steep liquor, soybean meal, soy protein, yeast. extract, meat extract and others. The nitrogen sources can be used singly or as a mixture.

 Inorganic salt compounds which may be included in the media include the chloride, phosphorus or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

 As sulfur source inorganic sulfur-containing compounds such as sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides but also organic sulfur compounds, such as mercaptans and thiols can be used.

 Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the phosphorus source.

 Chelating agents can be added to the medium to keep the metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.

 The fermentation media used according to the invention usually also contain other growth factors, such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine. Growth factors and salts are often derived from complex media components, such as yeast extract, molasses, corn steep liquor, and the like. In addition, suitable precursors can be added to the culture medium. The exact composition of the media compounds will depend heavily on the particular experiment and will be decided on a case by case basis. Information about the media optimization is available from the textbook "Applied Microbiol Physiology, A Practical Approach" (ed. P. M. Rhodes, P. F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media may also be obtained from commercial suppliers such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) and the like.

 All media components are sterilized either by heat (20 min at 1, 5 bar and 121 ° C) or by sterile filtration. The components can either be sterilized together or, if necessary, sterilized separately. All media components may be present at the beginning of the culture or added randomly or batchwise, as desired.

The temperature of the culture is usually between 15 ° C and 45 ° C, preferably 25 ° C to 40 ° C and can be kept constant or changed during the experiment. The pH of the medium should be in the range of 5 to 8.5, preferably around 7.0. The pH for cultivation can be controlled during cultivation by addition of basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid. To control the development of foam anti-foaming agents, such as. Fatty acid polyglycols ter, are used. To maintain the stability of plasmids, the medium can be selected selectively acting substances such. As antibiotics, are added. In order to maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, such. B. ambient air, registered in the culture. The temperature of the culture is usually 20 ° C to 45 ° C and. The culture is continued until a maximum of the desired product has formed. This goal is usually reached within 10 hours to 160 hours.

 The fermentation broth is then further processed. Depending on the requirement, the biomass can be wholly or partly by separation methods, such. As centrifugation, filtration, decantation or a combination of these methods are removed from the fermentation broth or completely left in it.

 Also, if the polypeptides are not secreted into the culture medium, the cells may be digested and the product recovered from the lysate by known protein isolation techniques. The cells may optionally be treated by high frequency ultrasound, high pressure, e.g. in a French pressure cell, by osmolysis, by the action of detergents, lytic enzymes or organic solvents, by homogenizers or by combining several of the listed methods.

 Purification of the polypeptides may be accomplished by known chromatographic techniques such as molecular sieve chromatography (gel filtration) such as Q-Sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, as well as other conventional techniques such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis. Suitable methods are described, for example, in Cooper, F.G., Biochemische Arbeitsmethoden, Verlag Walter de Gruyter, Berlin, New York or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.

 It may be advantageous to use for isolation of the recombinant protein vector systems or oligonucleotides which extend the cDNA by certain nucleotide sequences and thus encode altered polypeptides or fusion proteins, e.g. serve a simpler cleaning. Such suitable modifications are for example so-called "tags", such as anchors. the modification known as hexa-histidine anchors or epitopes that can be recognized as antigens of antibodies (described, for example, in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor (NY ) Press). These anchors may be used to attach the proteins to a solid support, e.g. a polymer matrix, which may for example be filled in a chromatography column, or used on a microtiter plate or on another carrier.

At the same time, these anchors can also be used to detect the proteins. In addition, conventional markers, such as fluorescence colorants, can be used to detect the proteins. Substances, enzyme markers which, after reaction with a substrate, form a detectable reaction product, or radioactive markers, used alone or in combination with the anchors for derivatization of the proteins. 7. Enzyme immobilization

The enzymes according to the invention can be used freely or immobilized in the enzymatic methods described herein. An immobilized enzyme is an enzyme which is fixed to an inert carrier. Suitable support materials and the enzymes immobilized thereon are known from EP-A-1 149849, EP-A-1 069 183 and DE-OS 100193773 and from the references cited therein. The disclosure of these documents is hereby incorporated by reference in its entirety. Suitable support materials include, for example, clays, clay minerals such as kaolinite, diatomaceous earth, perlite, silica, alumina, sodium carbonate, calcium carbonate, cellulose powders, anion exchange materials, synthetic polymers such as polystyrene, acrylic resins, phenolformaldehyde resins, polyurethanes and polyolefins such as polyethylene and polypropylene. The support materials are usually used to prepare the supported enzymes in a finely divided, particulate form, with porous forms being preferred. The particle size of the carrier material is usually not more than 5 mm, in particular not more than 2 mm (grading curve). Similarly, when using the dehydrogenase as a whole-cell catalyst, a free or immobilized form can be selected. Carrier materials are, for example, calcium alginate, and carrageenan. Enzymes as well as cells can also be cross-linked directly with glutaraldehyde (cross-linking to CLEAs). Corresponding and further immobilization processes are described, for example, in J. Lalonde and A. Margolin "Immobilization of Enzymes" in K. Drauz and H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol. III, 991-1032, Wiley-VCH, Weinheim ,

Experimental part:

 Unless otherwise specified, the cloning steps carried out in the context of the present invention, e.g. Restriction cleavage, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linkage of DNA fragments, transformation of microorganisms, growth of microorganisms, propagation of phage and sequence analysis of recombinant DNA using standard protocols, e.g. as in Sambrook et al. (1989) supra. described be performed.

1. Materials:

1.1 General

All chemicals were purchased from Sigma-Aldrich, Carl Roth and Biomol (Germany) and from Gerbu (Netherlands). All restriction endonucleases and T ₄ DNA ligase were purchased from ThermoScientific (Germany).

1.2 Media & Buffers:

LB medium containing tryptone 10 g, yeast extract 5 g, NaCl 10 g per liter of medium.

KPi buffer (50 mM, pH 8) containing 8.3 g K ₂ HP0 ₄ and 0.3 g KH ₂ P0 ₄ per liter buffer.

TEA buffer (100 mM, pH 7.5) containing 14.9 g of triethanolamine per liter of buffer, adjusted to pH 7.5 with 1 M HCl.

 Tris-HCl buffer (100 mM, pH 8.0, 8.5, 9.0) containing 12.1 g of tris (hydroxymethyl) aminomethane per liter of buffer, adjusted to the desired pH with 1 M NaOH. Citric acid buffer (100 mM pH 6) containing 2.4 g of citric acid monohydrate and 26.0 g of trisodium citrate dihydrate per liter of buffer.

 Digestion buffer containing 10 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl, pH 8.

 Wash buffer containing 20 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl, pH 8. Elution buffer containing 250 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl, pH 8.

For the use of lower molarity buffers, this was removed from the list (see above) with dest. Water diluted so that the desired molarity was obtained.

1.3 microorganisms: Table 1: Escherichia coli strains used

Strain genotype

Escherichia coli DH5a F-endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG O80dlacZAM15A (lacZYA-argF) U169, hsdR17 (rK -mK ⁺ ), A-

Escherichia coli BL21 (DE3) A7a-HSDH F- ompT gal to the ion hsdSB (rB-mB-) A (DE3

 [lad lacUV5-T7 gene 1 ind 1 sam7 nin5])

(7a-HSDH knock-out strain)

hshA-KanR ⁺

Escherichia coli strain DH5a (Novagen, Madison, Wisconsin, USA) was propagated at 37 ° C in LB medium containing appropriate antibiotics.

The Escherichia coli strain BL21 (DE3) A7a-HSDH was obtained from PharmaZell GmbH (see also WO201 1/147957).

1.4 Expression Vectors and Vector Constructs: For the expression of the recombinant dehydrogenases, the known expression vectors pET28a (+) (for the single expression of the genes) as well as pETDuet and pACYCDuet were used (Novagen, Madison, Wisconsin, USA) and the following vector constructs were generated:

PET28a (+) - 3a-HSDH (N): pET28a (+) - vector in which the gene of the 3a-HSDH from C. tes- tosteroni was cloned in the usual way via the Nde \ and Xho \ interfaces. This construct has an N-terminal 6xHis tag.

 PET28a (+) - 7ß-HSDH (C): pET28a (+) - vector in which the gene of 7ß-HSDH from C. aero-faciens was cloned in the usual way via the Nco \ and Xho \. This construct has a C-terminal 6xHis tag.

 • pDB02: pETDuet vector in which the gene of the 7ß-HSDH from C. aerofaciens via the interfaces Nco \ and Not \ in the 1. Multi-cloning site (MCS) and the 3a-HSDH from C. testosteroni via the interfaces Nde \ and Xho \ were cloned into the MCS2 in the usual way. This construct does not have a 6xHis tag.

PDB04: pETDuet vector in which the gene of glucose dehydrogenase from B.subtilis 168 via the Nco \ and Xho \ in the MCS1 and the 3a-HSDH from C. tes- tosteroni were intercepted in the usual way via the interfaces Nde \ and Xho \ in the MCS2. This construct does not have a 6xHis tag.

 • pDB04b: pETDuet vector in which the B. subtilis 168 glucose dehydrogenase gene is linked to the MCS1 via the Nco \ and Xho \ interstices and the C.-tes- tosteroni 3a-HSDH via the Nde \ and Xho \ cleavage sites MCS2 were eingkllo- in the usual way. QuikChange® PCR was used to introduce the mutation T188A into the gene of 3α-HSDH. This construct does not have a 6xHis tag.

 PA_7β-HSDH: pACYCDuet vector in which the gene of the 7ß-HSDH from C. aerofaciens was cloned in via the interfaces Λ / col and Xho in the usual way. This construct does not have a 6xHis tag.

 PA_7ß-HSDH [G39S / R64E]: pACYCDuet vector in which the gene of 7ß-HSDH from C. aerofaciens was cloned in the usual way via the interfaces Λ / col and Xho. By QuikChange® PCR, the mutations G39S and R64E were introduced into the gene of 7ß-HSDH This construct has no 6xHis tag.

 PA_GDH2: pACYCDuet vector in which the gene of B. subtilis 168 glucose dehydrogenase was cloned in the customary manner via the Bsa / NcoI and XhoI cleavage sites. This construct does not have a 6xHis tag.

The generated recombinant strains ("designer bugs") used for the reduction of 3,7-diketo-UDCA to UDCA are listed in Table 2.

 Table 2: Recombinant strains used. The strain Escherichia coli BL21 (DE3) A7a-HSDH was provided as an expression strain of PharmaZell. The plasmids used are listed in the chapter "Expression vectors and vector constructs"

Name plasmids

 E. coli DB02 pDB04 (GDH2 + 3a-HSDH) & pA_7β-HSDH

 E. coli DB03 pDB02 (7β-HSDH + 3a-HSDH) & pA_GDH2

 £. coli DB08 pDB04b (GDH2 + 3a-HSDH [T188A]) & pA_7β-HSDH [G39S / R64E]

2. Methods:

2.1 Cultivation

The cultivation took place in the shaking flask in LB medium at 37 ° C. At an OD ₆₀₀ = 0.6 - 0.8, gene expression was induced by the addition of 0.5 mM IPTG. Thereafter, the cells grew to harvest at 25 ° C for another 20 hours. 2.2 recovery of crude extract

The attracted cells were sonicated (25% cell suspension) and the recovered crude extract was separated from the cell debris by centrifugation (20,000 g, 30 min, 4 ° C).

2.3 Expression and Purification

E. coli βZ-27 (DE3) A7a-HSDH was transformed with the expression construct. For this purpose, the E. coli BL21 (DE3) A7a-HSDH strain containing the expression construct was incubated in LB medium with the corresponding antibiotic (50 μg ml kanamycin for pET28a, 36 μg ml chloramphenicol for pACYCDuet or 40 μg ml kanamycin + 29 μg ml Chloramphenicol for the whole cell biocatalyst). The cells were harvested by centrifugation (10,000xg, 30 min, 4 ° C). The pellet was resuspended in digestion buffer (25% cell suspension). The cells were disrupted under constant cooling by sonication for 2 minutes (30 W power, 10 - 25% working interval and 1 min break) using a Sonopuls HD2070 ultrasound machine (Bandelin, Germany). The digest was repeated three times. The cell extract was centrifuged (20,000xg, 30 min, 4 ° C). The supernatant was loaded onto a chromatography column (Thermo scientific, USA) previously equilibrated with 25 ml digestion buffer. Weak binding protein was removed by washing with 40 to 50 ml wash buffer. The fusion protein was eluted with elution buffer (10 ml). The procedure was carried out at RT. The eluate was concentrated by Centricon (ultrafiltration modules) and rebuffered to buffer 50 mM KPi (pH 8.0) by means of a PD10 column to remove the containing imidazole.

The protein concentration was determined by Bradford (see Method 2.5) (crude extract) or purified protein using Nanodrop © (ThermoScientific). In addition, the sample was analyzed by 15% SDS-PAGE and Coomassie Brilliant Blue staining.

2.4. standard conditions

2.4.1 Standard conditions for HSDH activity determination The reaction mixture contains a total volume of 1 ml: 880 μΙ 50 mM potassium phosphate (KPi) buffer, pH 8.0

100 μM 100 mM 3,7-diketo-UDCA (dissolved in 50 mM KPi, pH 8) 10 μΙ enzyme solution (diluted in buffer, in the range of 1 to 5 U / ml)

10 μΙ 50 mM NAD ⁺ or NADP ⁺ (dissolved in ddH ₂ 0)

The decrease in absorbance is measured at 340 nm over a period of 30 seconds and the activity is calculated as the enzyme unit (U, ie μη "ΐοΙ / η" ΐίη) using the molar extinction coefficient of 6.22 mM ^"1 xcm ^{" 1} .

2.4.2. Standard conditions for the activity determination of glucose dehydrogenase

The reaction mixture contains a total volume of 1 ml: 940 μΙ 50 mM potassium phosphate (KPi) buffer, pH 8.0

40 μΙ 2.5 M glucose (dissolved in distilled water)

10 μΙ enzyme solution (diluted in buffer, in the range of 1 to 5 U / ml)

10 μΙ 50 mM NAD ⁺ or NADP ⁺ (dissolved in ddH ₂ 0)

The increase in absorbance is measured at 340 nm over a period of 30 seconds and the activity is expressed as the enzyme unit (U, ie μη "ΐοΙ / η" ΐίη

2.5 Protein determination using Bradford

The samples (100 μΙ each) were mixed with 900 μΙ Bradford reagent and incubated for at least 15 min in the dark. The protein content was determined at 595 nm against a calibration curve (BSA) in the concentration range of the assay used.

2.6 Molecular biological working methods

Molecular biological work is done, as far as no other information is given, based on established methods, eg described in: Sambrook, J., Fritsch, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990). 2.7 High-performance liquid chromatography (HPLC)

For the qualitative and quantitative analysis of all bile acids involved in the reactions considered, the HPLC system Jasco was used, consisting of the interface LC-Net II / ADC, 3-way degasser DG2080-53, autosampler AS2059SFPIus, two pumps PU-2080Plus, column oven CO-2060Plus, RI detector RI-2031 Plus and multi-wavelength detector MD-2010Plus. The substances were separated using a Purospher STAR® RP-18e (5 μηη) reversed-phase chromatography column from Merck (Darmstadt, Germany). A gradient elution was selected in which the mobile phase used was a mixture of acetonitrile and phosphoric acid water (pH 2.6). The latter served to obtain a uniform degree of protonation. The analysis of the substances was carried out at 25 ° C oven temperature and the detection was carried out with a UV sensor at λ = 200 nm.

The gradient used was: 0-15 min linear increase from 20% acetonitrile to 35%, 15-53 min linear increase to 70% acetonitrile, 53-75 min constant 70% acetonitrile, 75-78 min linear decrease to 20% acetonitrile, 78 - 88 min constant 20% acetonitrile.

3. Examples

Example 1: Chemical oxidation of CDCA 1.1 SWERN oxidation

In a 1 L three-necked flask, oxalyl chloride (70 mmol, 6.02 ml) is dissolved in about 50 ml CH ₂ Cl ₂ and cooled to -78 ° C. with an acetone / T rock ice bath. A solution of dimethyl sulphoxide (130 mmol, 9.24 ml) in 15 ml CH ₂ Cl _{2 is} added dropwise via a dropping funnel for 5 min. A solution of chenodeoxycholic acid (CDCA) (30 mmol, 1 L, 78 g) in a mixture of CH ₂ Cl ₂ / DMSO (total of 30 mL [about 15 mL DMSO, balance CH ₂ Cl ₂ ]) is added via another Dropping funnel added within 5 min. The reaction mixture is stirred for 15 min. stirred, then triethylamine (150 mmol, 21 ml 42 ml) is added, stirred for a further 5 min and the cold bath removed. To the reaction mixture heated to RT, 180 ml of dist. H ₂ 0 and stirred for 5 min. Subsequently, aqueous and organic phases are separated and the aqueous phase is extracted with 200 ml of CH ₂ Cl ₂ . The combined organic phases are successively with 250 ml of 0.5 M HCl and dist. H ₂ 0, dried over NaS0 ₄ and freed from the solvent in vacuo.

Reaction:

 Chenodeoxycholic acid 3,7-diketo-UDCA

 Scheme 1: Reaction scheme of the chemical oxidation of CDCA using the SWERN protocol.

The product was obtained with a yield of 46% and a purity of about 90%.

1 .2 Tempo oxidation

In a 100 ml two-necked flask with internal thermometer CDCA (300 mg, 764 μηηοΙ) was dissolved in 20 ml of acetonitrile. After cooling the reaction solution to 4 ° C, sodium bromide (15.7 mg, 153 mol) and TEMPO (2,2,6,6-tetramethylpiperidinyloxy) (4.8 mg, 30.6 mol) were added. Subsequently, a precooled NaOCl solution (10.0 ml, 0.76 M, pH = 8.8) in aliquots a 1 .0 ml was added with constant stirring within 5 minutes to the reaction solution, wherein the internal temperature was kept below 5 ° C. After a reaction time of 60 minutes, a conversion control was carried out by TLC (cyclohexane / ethyl acetate / acetic acid 1: 1: 0.005, v / v) and the reaction mixture was brought to room temperature. 20 ml of 1 M HCl was added, which dissolved any solid and then extracted twice with 50 ml of ethyl acetate. The combined extracts were washed with 30 ml of 0.5 M HCl, dried over MgS0 ₄ and freed from the solvent. 3,7-diketo-UDCA was obtained as a colorless solid.

Reaction:

 Chenodeoxycholic acid 3.7-diketo-UDCA

 Scheme 2: Reaction scheme of chemical oxidation of CDCA by TEMPO.

The product was obtained with a yield of 78% and a purity of about 95%.

1 .3 sodium hypochlorite / sodium bisulfite

The chenodeoxycholic acid (10 g, 24.5 mmol) was dissolved in a 500 ml three-necked flask in acetic acid (1 x 10 ml) at 18 ° C. Hypochlorite (35 ml, 2.66 eq, 65.15 mmol) was added over 40 minutes to the resulting brown solution via a dropping funnel. The temperature was kept at 7 -15 ° C. The milky suspension was then stirred for a further three hours with cooling. To neutralize the excess hypochlorite, sodium bisulfite solution (20.4 mmol, 35 ml) was added dropwise thereto. The temperature was kept constant at 15 ° C again. The mixture was stirred for a further 45 min, during which the initially clear solution became slightly milky again. After addition of H ₂ O (160 ml), a milky suspension was obtained. This milky suspension was left for 45 min. heated to 70 ° C, then it was cooled down to 20 ° C within one hour. The product was filtered through a suction pad and washed with 600 ml of water until no more acetic acid odor was heard. The product was dried for several days in the desiccator and finally on the Schlenkin. The product was obtained with a yield of 7.97 g (79.7%) and a purity of 98.6%. The purity was determined by means of ¹ H-NMR and HPLC.

Reaction:

 Chenodeoxycholic acid 3,7-diketo-UDCA

Scheme 3: Reaction scheme of the chemical oxidation of CDCA with stoichiometric amounts of NaOCl. 1.4 Analytics:

After each chemical oxidation, the analysis was carried out with NMR (C13 & H) and HPLC.

¹ H-NMR (500 MHz, DMSO): δ [ppm] = 1.96 (s, 1H), 2.90 (dd, 1H, J = 12.7 Hz, 5.5 Hz), 2.54 (t, 1H, J = 1 1.4 Hz), 2.35 (dt, J = 14.7 Hz, 5.2 Hz), 2.26-2.04 (m, 5H), 2.00-1 .92 (m, 5H), 1 .86-1 .64 (m, 3H) , 1 .56-1.34 (m, 5H), 1 .28-1 .15 (m, 6H), 1 .07 (q, 1 H, J = 9.5 Hz), 0.99-0.91 (m, 1 H), 0.88 (d, 3H, J = 6.5 Hz), 0.64 (s, 3H).

MS (ESI): m / z = 41 1 .0 ([M + Na] ⁺ ), 445.0 ([M + K] ⁺ ), 799.5 ([2M + Na] ⁺ ).

RP-HPLC = CDCA = 31, 1 min .; 3,7-diketo-UDCA = 29.7 min.

Example 2: QuikChange PCR

QuikChange® PCR (QC-PCR) was used to target the exchange of individual amino acids. The primers used are shown in Table 3, which effected the desired exchange at positions 39 and 64 for the 7ß-HSDH and position 188 for the 3a-HSDH, respectively.

Table 3: Primers used for QuikChange® PCR.

The reaction was first followed by a 2 min initial denaturation step at 95 ° C. Thereafter, 23 cycles of denaturation (30 sec. At 95 ° C), primer hybridization and elongation (2.5 min. At 72 ° C) were performed. The final step was a final elongation of 72 ° C for 10 minutes before the polymerase chain reaction was terminated by cooling to 15 ° C. Table 4 indicates the corresponding composition of the reaction mixture.

Table 4: PCR approach for the generation of the various 3α and 7β-HSDH variants

The template used was a pET28a vector with the corresponding wild-type gene. In order to exchange amino acids in protein sequences targeted, the DNA sequence of the corresponding gene is mutated positionally. For this purpose, each other used complementary primers that carry the desired mutation in their sequence. The template is N6-adeninmethylierte, double-stranded plasmid DNA carrying the gene to be mutated. N6-adenine methylated plasmid DNA is obtained from the dam ⁺ £. co // strain isolated, such as E. coli DH5a. The polymerase chain reaction is carried out as described above. The primers are extended complementarily to the template, resulting in plasmids with the desired mutation, which have a strand break. Unlike other PCR reactions, the DNA yield only increases linearly, since newly formed DNA molecules can not serve as templates for the PCR reaction. After completion of the PCR reaction, the PCR product was purified by means of the PCR purification kit (Analytik Jena) and the parent N6-adenine-methylated DNA was digested using the restriction enzyme dpn \. This enzyme has the peculiarity that it nonspecifically restricts N6 adenine methylated DNA, but not the newly formed, unmethylated DNA. The restriction was carried out by adding 1 μΙ dpn \ to the purified PCR reaction mixture and incubating for at least 2 h or overnight at 37 ° C.

8 μΙ of this approach were used to transform 100 μΙ of chemically competent DH5o cells.

Example 3: Biochemical Characterization of 3α and 7β-HSDH In order to determine the enzyme kinetic parameters of both the ββ and 3a-HSDH, the genes of the 7β-HSDH mutants were analyzed using the plasmid pET28a_7β HSDH (C) and pET28a_3α, respectively. HSDH (N) expressed in E. coli BL21 (DE3) A7a-HSDH. These variants have a C-terminal (7ß HSDH) and N-terminal (3a-HSDH) 6xHis tag, respectively. The crude extract of the disrupted cells was purified by means of Ni-NTA (see Section 2.3) and to have an SDS page for the control of the expression as well as the purification (see FIG. 1).

From Figure 1 shows that the expression was successful, it can be seen in both cases, marked overexpression bands. Furthermore, almost completely foreign protein could be removed by the Ni-NTA purification. In the measurements for determining the kinetic constants for the 3a-HSDH different substrate concentrations between 10 μΜ and 10 mM for 3,7-diketo-UDCA and for the intermediate 3-keto-UDCA at a constant 0.2 mM NADH were used. The concentration of 3,7-diketo-UDCA was determined to determine the constants for the cofactor kept constant at 1 mM and examined the NAD (P) H concentration between 10 μΜ and 0.30 mM. FIG. 2 shows the progress diagrams as well as the values in Table 5.

For the determination of the kinetic constants for the 7ß-HSDH different substrate concentrations between 10 μΜ and 15 mM for 3,7-diketo-UDCA as well as for the intermediate 7-KLCA between 10 μΜ and 12,5 mM at constant 0.2 mM NADPH used. For the determination of the constants for the cofactor, the concentration of 3,7-diketo-UDCA was kept constant at 1 mM, for the cosubstrate 7-KLCA constant at 5 mM, and the NADPH concentration was examined between 10 μΜ and 0.3 mM , FIG. 3 shows the progress diagrams and the corresponding values in Table 5.

Table 5: Kinetic constants of 3a- and 7ß-HSDH for the substrate 3,7-diketo-UDCA as well as 3-keto-UDCA or 7-KLCA as well as the associated cofactor. In each case, the compound indicated in the "Substrate" column was varied: in the cosubstrate column, the concentration of NAD (P) H was 0.2 mM and of 3,7-diketo-UDCA 0.1 mM (3a-HSDH ) or 10 mM (7β-HSDH) X = no inhibition.

From Table 5 it can be seen that of the two enzymes 3a-HSDH and 7ß-HSDH only the 3a-HSDH with the diketone 3,7-diketo-UDCA and also with the monoketone 3-keto-UDCA has a substrate inhibition. For the monoketone, however, the inhibition is less pronounced. Furthermore, it is conspicuous that the 7ß-HSDH has no inhibition. The comparison of both enzymes shows that the 3a-HSDH despite the substrate inhibition has a higher activity than the 7 ß-HSDH. Example 4: Biotransformations of 3,7-diketo-UDCA to UDCA

For the synthesis of UDCA from 3,7-diketo-UDCA, preparations were made with both crude extract (see Section A below) and whole cell biocatalysts (see Section B, below)). The concentration of 3,7-diketo-UDCA was 10 mM (with crude extract) or 40 mM (whole cell biocatalysts).

A) Biotransformation with crude extract

Conditions: 0.5 mg 3a-HSDH (N); 0.5 mg 7-βHSDH (C); 5 mg GDH; 10mM 3,7-diketo-UDCA; 0.5 mM NAD ⁺ as well as 0.5 mM NADP ⁺ ; 50 mM glucose in 100 mM buffer (TEA, pH 7.5, Tris-HCl, pH 8.0, Tris-HCl, pH 8.5, Tris-HCl, pH 9.0). After 16 hours, a sample was taken and analyzed by HPLC.

From Figure 4 it can be seen that the reaction is highly pH-dependent: After 16 hours, neither starting material nor the intermediate compound 3-keto-UDCA are detectable in all batches. Furthermore, it can be seen that at a pH of 7.5 still about 49% of the intermediate product 7-KLCA is detectable and at a pH of 8 only about 14%. In the reactions with a pH of 8.5 and 9.0, a complete reaction could be measured.

B) Biotransformation with whole-cell biocatalysts

B1) whole cell biocatalyst £. coli DB03: Conditions:

5 mg ₍ BFM ₎ / ml E. coli DB03; 40 mM 3,7-diketo-UDCA; 0.2 mM NAD ⁺ as well as 0.2 mM NADP ⁺ ; 200mM glucose in 5mM buffer (citric acid, pH 6.0). The reaction was carried out on pH stat / titrino, the pH being kept constant at 6.0 by the addition of 10% NaOH. After 0, 5 and 20.5 hours, a sample was taken and analyzed by HPLC.

FIG. 5 shows that after 20.5 hours, a conversion of about 90% could be detected with the whole-cell biocatalyst E. coli DB03. Furthermore, 10% intermediate 7-KLCA could be detected. The intermediate 3-keto-UDCA accumulated after 5 hours with a share of about 30%, but it was subsequently fully implemented by the 3a-HSDH.

B2) whole-cell biocatalyst £. coli DB02:

Conditions:

5 mg ₍ BFM ₎ / ml E. coli DB02; 40 mM 3,7-diketo-UDCA; 0.2 mM NAD ⁺ as well as 0.2 mM NADP ⁺ ; 200mM glucose in 5mM buffer (citric acid, pH 6.0). The reaction proceeded on pH stat / titrino, the pH was kept constant at 6.0 by the addition of 10% NaOH. After 0, 5 and 20.5 hours, a sample was taken and analyzed by HPLC.

From Figure 6 shows that after 27 hours, a conversion of about 93% with the whole-cell biocatalyst E. coli DB03 could be detected. The remaining 7% are assigned to the intermediate 7-KLCA. The intermediate 3-keto-UDCA could not be detected at the times taken.

attachment

Amino acid sequence of NADH-dependent 3a-HSDH from C. testosteroni (28.56 kDa, with N-terminal His tag [underlined]); MGSSHHHHHHSSGLVPRGSHMSIIVISGCATGIGAATRKVLEAAGHQIVGIDIRDAEVI- ADLSTAEGRKQAIADVLAKCSKGMDGLVLCAGLGPQTKVLGNVVSVNYFGATELMDAFL- PALKKGHQPAAVVISSVASAHLAFDKNPLALALEAGEEAKARAIVEHAGEQGGNLAYAGS- KNALTVAVRKRAAAWGEAGVRLNTIAPGATETPLLQAGLQDPRYGESIAKFVPPM- GRRAEPSEMASVIAFLMSPAASYVHGAQIVIDGGIDAVMRPTQF

Amino acid sequence of NADPH-dependent 7ß-HSDH from C. aerofaciens (29.82 kDa, with C-terminal 6xHis tag [underlined])

MNLREKYGEWGLILGATEGVGKAFCEKIAAGGMNWMVGRREEKLNVLAGEIRETYG- VETKWRADFSQPGAAETVFAATEGLDMGFMSYVACLHSFGKIQDTPWEKHEAMIN- VNWTFLKCFHHYMRIFAAQDRGAVINVSSMTGISSSPWNGQYGAGKAFILKMTEAVACE- CEGTGVDVEVITLGTTLTPSLLSNLPGGPQGEAVMKIALTPEECVDEAFEKLGKELSVIAG- QRNKDSVHDWKANHTEDEYIRYMGSFYRDLEHHHHHH

Amino acid sequence of NAD (P) H-dependent glucose dehydrogenase from B. subtilis strain 168 (28.07 kDa); underlined twice are the amino acids that were exchanged.

MYPDLKGKWAITGAASGLGKAMAIRFGKEQAKVVINYYSNKQDPNEVKEEVIKAG- GEAWVQGDVTKEEDVKNIVQTAIKEFGTLDIMINNAGLENPVPSHEMP- LKDWDKVIGTNLTGAFLGSREAIKYFVENDIKGNVINMSSVHEVIPWPLFVHYAASKG- GIKLMT TLALEYAPKGIRVNNIGPGAINTPINAEKFADPKQKADVESMIPMGYIGE- PEEIAAVAAWLASKEASYVTGITLFADGGMTLYPSFQAGRG Sequences used herein

 AS = amino acid sequence

NS = nucleic acid sequence

Reference is expressly made to the disclosure of the references mentioned herein.

Claims

claims Process for the preparation of a ursodeoxycholic acid (UDCA) of the formula (1)

wherein R is alkyl, H, an alkali metal ion or N (R ³ ) ₄ ⁺ in which the radicals R ^{3 are} identical or different and are H or alkyl, or where the group -C0 ₂ R is represented by an acid amide group of the formula ONR ¹ R ^{2 is} replaced, in which R ¹ and R ² independently of one another are an alkyl radical, a) a chenodeoxycholic acid (CDCA) of the formula (2)

wherein R or the group -C0 ₂ R have the meanings given above, to a 3,7-diketo-ursodeoxycocholic acid (3,7-diketo -UDCA) of the formula (3)

wherein R or the group -C0 ₂ R have the meanings given above, chemically oxidized; and optionally purifying the formed 3,7-deco-UDCA, b) the 3,7-diketo-UDCA of formula (3) formed in step a) in the presence of at least one 7β-hydroxysteroid dehydrogenase (7β-HSDH) and in the presence of at least one 3a-hydroxysteroid dehydrogenase (3a-HSDH) to UDCA compound of the above formula (1) enzymatically reduced, and optionally further purified the reaction product. 2. The method of claim 1, wherein subjecting in step a) a chemical oxidation, wherein the oxidizing agent used is capable of oxidizing a secondary hydroxyl group. 3. The method of claim 2, wherein the oxidation step is selected from SWERN oxidation and TEMPO oxidation and sodium hypochlorite oxidation. 4. The method according to any one of the preceding claims, wherein one carries out stage b) in the presence and consumption of reduction equivalents (NADH and / or NADPH). 5. The method according to any one of the preceding claims, wherein the 3a-HSDH and 7ß-HSDH used in step b) have the same or different cofactor specificity. 6. The method according to any one of the preceding claims, wherein one couples step b) for cofactor regeneration with at least one further, regenerating the spent regeneration enzyme. 7. The method of claim 6, wherein spent NADPH is regenerated by coupling with an NADPH regenerating enzyme, which is especially selected from NADPH dehydrogenases, alcohol dehydrogenases (ADH), and NADPH regenerating formate dehydrogenases (FDH) and an NADPH regenerating glucose dehydrogenase (GDH) and NADPH-regenerating glucose-6-phosphate dehydrogenases (G6PDH); and / or where spent NADH is regenerated by coupling with an NADH-regenerating enzyme, which is especially selected from NADH dehydrogenases, NADH regenerating formate dehydrogenases (FDH), NADH regenerating alcohol dehydrogenases (ADH), NADH regenerating glucose-6 Phosphate dehydrogenases (G6PDH), NADH-regenerating phosphite dehydrogenases (PtDH) and NADH-regenerating glucose dehydrogenases rogenases (GDH); and / or wherein spent NADH and / or spent NADPH is regenerated by coupling with a cofactor regenerating enzyme selected from a GDH. 8. The method of claim 7, wherein spent NADH and / or spent NADPH is regenerated by coupling to a both cofactor-regenerating thermostable mutant of a Bacillus subtilis GDH, in particular the E170K, Q262L double mutant of SEQ ID NO: 14J 9. The method of claim 7 or 8, wherein adding glucose to the reaction medium as a substrate for the GDH. 10. The method of claim 9, wherein the reaction medium addition NAD ⁺ and / or NADP ⁺ added to (additional) reduction equivalents for the enzymatic conversion to the 3,7-deco-UDCA compound of the formula (3) according to step b) to generate. 1 1. The method according to any one of the preceding claims, wherein the step b) with isolated or enriched enzymes (7ß-HSDH, the 3a-HSDH and optionally the enzyme required for cofactor regeneration) or with crude cell extract (containing the 7ß- HSDH, the 3α-HSDH and optionally the enzyme needed for cofactor regeneration) or with recombinant microorganisms which functionally express the 7β-HSDH, the 3α-HSDH and, if necessary, the enzyme required for cofactor regeneration. 12. The method of claim 1 1, wherein the one uses a recombinant microorganism in which unwanted side activity of a 7a-HSDH is inhibited. 13. The method according to any one of the preceding claims, wherein the 7ß-HSDH is selected from the following enzymes and enzyme mutants, wherein the non-mutated enzyme has an amino acid sequence according to SEQ ID NO: 1 1: Position of the type Preferred mutation Cofactor mutation usage WT (Colinsella WT No NADPH aerofaciens)  C-His-tag WT 6xHis tag NADPH N-His tag  T17 s T17 F, A, I, or S NADPH /  NADH  G39 s G39 S, A, D, V, I, L, C, K, Y, F, R, T, P, N, E, Q, H, or NADPH /  W NADH  R64 s R64 E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I, Y, H or N NADPH G39 / R64 D G39S / R64E or G39S / R64D NADPH G39 / R40 D G39D / R40F, G39D / R40I, G39D / R40L, or G39D / R40W, NADH G39 / R40 / R41 T G39D / R40F / R41 (K, Q, S or R) NADH  G39D / R40I / R41 N  G39 / R40 / R41 / K44 Q G39D / R40F / R41 K / K44 (G, N or Q) NADH R40 S R40D, E, I, V, L, G or A. NADH R41 S R41 N.I.L or V NADH G39 / R40 R40 / R41 D G39D / R40I, G39D / R40V, G39D / R40L or R40D / R41 I NADH G39 / R41 WT = wild type; S = single mutation; D = double mutation; T = triple mutation; Q = quadruple mutation Included here are variants which are extended C- and / or N-terminal, in particular with a His-tag sequence; and wherein the above mutants can additionally be mutated in each of the positions K44, R53, K61, R64. 14. The method according to any one of the preceding claims, wherein the 3a-HSDH is selected from the following enzymes and enzyme mutants, wherein the non-mutated enzyme has an amino acid sequence according to SEQ ID NO: 8. Position of the type Preferred mutation cofactor  Mutation usage WT (Coma-WT NADH  monas testosteroni)  C-His-Tag WT 6xHis-Tag NADH  N-His-tag?  R34 s R34N, L, S or C NADH  L68 * s L68A, C, K, S or V NADH  R34 / L68 D R34N / L68A NADH P185 * S P185A, G, T, V or S NADH  T188 * S T188A, S, V, or G NADH T188 / P185 D P185A / T188A; NADH  P185A / T188S;  L68 / P185 D L68A / P185A and L68C / P185A NADH L68 / T188 D L68A / T188A and L68A / T188S; NADH  L68C / T188A and L68C / T188S WT = wild type; S = single mutation; D = double mutation  Included here are mutants which are extended C- and / or N-terminal, in particular with a His-tag sequence. 15. The method according to any one of the preceding claims, wherein the following enzyme combinations are used

 WT = wild type