US20140087421A1 - NOVEL 7Beta-HYDROXYSTEROID DEHYDROGENASE MUTANTS AND PROCESS FOR THE PREPARATION OF URSODEOXYCHOLIC ACID - Google Patents

NOVEL 7Beta-HYDROXYSTEROID DEHYDROGENASE MUTANTS AND PROCESS FOR THE PREPARATION OF URSODEOXYCHOLIC ACID Download PDF

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US20140087421A1
US20140087421A1 US13/993,235 US201113993235A US2014087421A1 US 20140087421 A1 US20140087421 A1 US 20140087421A1 US 201113993235 A US201113993235 A US 201113993235A US 2014087421 A1 US2014087421 A1 US 2014087421A1
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hsdh
mutant
fdh
enzyme
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Dirk Weuster-Botz
Michael Braun
Arno Aigner
Boqiao Sun
Christina Kantzow
Sven Bresch
Daniel Bakonyi
Werner Hummel
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Pharmazell GmbH
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    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
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    • C12P33/00—Preparation of steroids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P33/00—Preparation of steroids
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    • C12P33/00—Preparation of steroids
    • C12P33/06—Hydroxylating
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01201—7-Beta-hydroxysteroid dehydrogenase (NADP+) (1.1.1.201)

Definitions

  • the invention relates to novel 7 ⁇ -hydroxysteroid dehydrogenase mutants, to the sequences that code for these enzyme mutants, to processes for the preparation of the enzyme mutants and use thereof in enzymatic reactions of cholic acid compounds, and especially in the preparation of ursodeoxycholic acid (UDCA); the invention also relates to novel processes for the synthesis of UDCA using the enzyme mutants; and to the preparation of UDCA using recombinant, multiply-modified microorganisms.
  • ursodeoxycholic acid and the related diastereomer chenodeoxycholic acid (CDCA), among others, have been used for many years for the drug treatment of gallstone disease.
  • the two compounds differ only in the configuration of the hydroxyl group on carbon atom 7 (UDCA: ⁇ -configuration, CDCA: ⁇ -configuration).
  • UDCA ursodeoxycholic acid
  • CDCA chenodeoxycholic acid
  • a serious disadvantage is, among other things, the following: as the chemical oxidation is not selective, the carboxyl group and the 3 ⁇ and 7 ⁇ -hydroxyl group must be protected by esterification.
  • the 12 ⁇ -HSDH oxidizes CA selectively to 12-keto-CDCA.
  • the two protection steps required according to the classical chemical method are then omitted.
  • the CA is first oxidized from 7 ⁇ -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 12 ⁇ -HSDH to 7,12-diketo-LCA. These two enzymes are each NADH-dependent. After reduction by 7 ⁇ -HSDH (NADPH-dependent) from Clostridium absonum ATCC 27555 (DSM 599) (MacDonald, I. A. and P. D.
  • the applicant's earlier international patent application PCT/EP2010/068576 describes a novel 7 ⁇ -HSDH from Collinsella aerofaciens ATCC 25986, which among other things has a molecular weight (in SDS-gel electrophoresis) of about 28-32 kDa, a molecular weight (in gel filtration, in nondenaturing conditions, such as in particular without SDS) from about 53 to 60 kDa, and the capacity for stereoselective reduction of the 7-carbonyl group of 7-keto-LCA to a 7 ⁇ -hydroxyl group.
  • a molecular weight in SDS-gel electrophoresis
  • a molecular weight in gel filtration, in nondenaturing conditions, such as in particular without SDS
  • DHCA is reduced by the pair of enzymes 7 ⁇ -HSDH and 3 ⁇ -HSDH individually in succession or in one pot to 12-keto-UDCA.
  • UDCA can therefore be synthesized from CA in just three steps. 7 ⁇ -HSDH is dependent on the cofactor NADPH, whereas 3 ⁇ -HSDH requires the cofactor NADH.
  • the availability of pairs of enzymes with dependence on the same cofactor or extended dependence would be advantageous, because this could simplify cofactor regeneration.
  • the problem to be solved by the invention is to provide further improved 7 ⁇ -HSDHs.
  • enzyme mutants should be provided, which can be used even more advantageously for enzymatic or microbial preparation of UDCA via the stereospecific reduction of DHCA in 7-position to 3,12-diketo-7 ⁇ -CA, and in particular have reduced substrate inhibition and/or have altered cofactor usage (increased, altered specificity or extended dependence).
  • Another problem is to provide novel enzymatic and microbial synthesis routes, which in particular are also characterized by simplified cofactor regeneration in the reductive preparation of UDCA via DHCA.
  • biocatalytic (microbial or enzymatic) process comprising the enzymatic conversion of DHCA via two partial reductive steps catalyzed by 7 ⁇ -HSDH or 3 ⁇ -HSDH, which can take place simultaneously or with a time delay in any order, to 12-keto-UDCA and cofactor regeneration using dehydrogenases, such as in particular formate dehydrogenase (FDH) enzymes or glucose dehydrogenase (GDH) enzymes, which regenerate the spent cofactor from the two partial reductive steps.
  • dehydrogenases such as in particular formate dehydrogenase (FDH) enzymes or glucose dehydrogenase (GDH) enzymes, which regenerate the spent cofactor from the two partial reductive steps.
  • FIG. 1 a shows the amino acid sequence of 7 ⁇ -HSDH from Collinsella aerofaciens and FIG. 1 b shows the coding nucleic acid sequence for the amino acid sequence of FIG. 1 a ;
  • FIG. 1 c shows the amino acid sequence of 3 ⁇ -HSDH from Comanomonas testosteroni and FIG. 1 d shows the coding nucleic acid sequence for the amino acid sequence of FIG. 1 c ;
  • FIG. 1 e shows the amino acid sequence of 3 ⁇ -HSDH from Rattus norvegicus and
  • FIG. 1 f shows the coding nucleic acid sequence for the amino acid sequence of FIG. 1 e ;
  • FIG. 1 g shows the coding nucleic acid sequence of the FDH mutant D221G and
  • FIG. 1 h shows the amino acid sequence for the nucleic acid sequence of FIG. 1 g.
  • FIG. 2 shows the SDS-gel of a purified 7 ⁇ -HSDH prepared according to the invention, with, on lane 1: cell raw extract; lane 2: purified protein; lane M: Page RoulerTM, molecular weight marker (Fermentas, Germany).
  • FIG. 3 shows the construction scheme of (A) pET21a(+), (B) pET22b(+), (C) pCOLA (Mod) and (D) pET28a(+).
  • FIG. 4 shows the construction schemes of pET21a(+) FDH D221G ( FIG. 4 a ) and pET21a(+) FDH 7 ⁇ -HSDH ( FIG. 4 b ).
  • FIG. 5 shows the construction scheme of pCOLA(mod) 3 ⁇ -HSDH.
  • FIG. 6 shows an activity comparison for / ⁇ -HSDH wild type and the 7 ⁇ -HSDH mutants G39A and G39S, namely in row A: the plot of the specific enzyme activity versus different substrate concentrations used at constant cofactor concentration of 100 ⁇ M; and in row B the plot of enzyme activity versus different cofactor concentrations used at constant substrate concentration of 0.3 mM.
  • FIG. 7 shows the HPLC chromatogram of the biotransformation of DHCS with 7 ⁇ -HSDH and 3 ⁇ -HSDH in the whole-cell process. It shows the HPLC chromatogram of the whole-cell conversion of the strain E. coli BL21 (DE3) hdhA ⁇ KanR + pET21a(+) FDH 7 ⁇ -HSDH pCOLA(mod) 3 ⁇ -HSDH.
  • the starting conditions selected were 50 mM substrate, 400 mM cosubstrate, pH 6.0. Sampling was carried out after 48 h.
  • the peaks of 12-keto-UDCA (1), an unknown by-product (2), 3,12-diketo-UDCA (3), 7,12-diketo-UDCA (4) and DHCA (5) can be seen.
  • FIG. 8 shows the construction scheme of the triple vector pET21a(+) FDH 7beta (G39A) that bears the coding sequences for FDH D221G, 7 ⁇ -HSDH G39A and 3 ⁇ -HSDH.
  • FIG. 9 shows the course of whole-cell biotransformation of DHCA.
  • the proportionate peak areas (according to HPLC analysis) of 12-keto-UDCA, 3,12-diketo-UDCA, 7,12-diketo-UDCA and DHCA are shown as a function of time.
  • FIG. 10 shows a sequence comparison between the 7 ⁇ -HSDH wild type and selected mutants.
  • the sequences designated as “7beta-HSDH wild type”, “7beta-HSDH G39D”, “7beta-HSDH G39D R40L”, “7beta-HSDH G39D R40I” and “7beta-HSDH G39D R40V” correspond to SEQ ID NO:2, SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39.
  • FIG. 11 shows an enzyme-kinetic investigation of 7 ⁇ -HSDH and the mutants thereof.
  • the specific enzyme activity is plotted versus different substrate concentrations used (DHCA concentration) at a constant cofactor concentration of 0.1 mM NADPH or 0.5 mM NADH.
  • FIG. 12 shows a schematic representation of a two-step enzymatic reduction of dehydrocholic acid to 12-keto-ursodeoxycholic acid according to the invention, wherein an NADH-dependent 7 ⁇ -HSDH is used and a formate dehydrogenase (FDH) is used for cofactor regeneration.
  • FDH formate dehydrogenase
  • FIG. 13 shows a schematic representation of the principle of construction of the vectors pFr7(D), pFr7(DI), pFr7(DL) and pFr7(DV) that were used for the expression of various NADH-dependent mutants of 7 ⁇ -HSDH.
  • FIG. 14 shows the proportions of bile salts in biotransformation batches using NADH-dependent 7 ⁇ -HSDH mutants. Results after 24 h process time and using 100 mM substrate (DHCA) are shown.
  • FIG. 15 shows a schematic representation of the vector pFr3T7(D).
  • FIG. 16 shows the result of a whole-cell biotransformation with the strain E. coli BL49 pFr3T7(D), which comprises the NADH-dependent 7 ⁇ -HSDH (G39D), an NADH-dependent 3 ⁇ -HSDH and an NADH-dependent FDH.
  • the biotransformations were carried out in the following conditions: 20 mL reaction volume, 17.7 g/l BTM cells, 100 mM DHCA, 500 mM ammonium formate, 26% glycerol, 50 mM MgCl 2 , 50 mM KPi buffer (pH 6.5). During the first 5 hours the pH was adjusted manually with formic acid to the initial value at hourly intervals.
  • FIG. 17 shows schematic representations of various vectors used: a) pF(G)r7(A)r3 (wherein this corresponds to the vector shown in FIG. 8 ) and b) pF(G)r7(S)r3.
  • FIG. 18 shows a schematic representation of the two-step enzymatic reduction of dehydrocholic acid to 12-keto-ursodeoxycholic acid, wherein an NADPH-dependent 7 ⁇ -HSDH is used and a formate dehydrogenase (FDH) mutant, which regenerates both NADPH- and NADH, is used for cofactor regeneration.
  • FDH formate dehydrogenase
  • FIG. 19 shows a time-resolved variation of the biotransformation with the strain E. coli BL49 pF(G)r7(A)r3 at the liter scale.
  • the batch contained 70 mM DHCA, 17.7 g/l BTM of the stored biocatalyst, 500 mM sodium formate, 26% (v/v) glycerol, 50 mM MgCl 2 , in 50 mM KPi buffer (pH 6.5). Using pH adjustment, the pH was maintained at pH 6.5 throughout the biotransformation.
  • FIG. 20 shows a schematic representation of the vector p3T7(A)rG.
  • FIG. 21 shows a schematic representation of the vector p7(A)T3rG.
  • FIG. 22 shows a time-resolved variation of the biotransformation with the strain E. coli BL49 p7(A)T3rG, which comprises a GDH for cofactor regeneration.
  • the batch contained 100 mM DHCA, 17.7 g/l BIM of the stored biocatalyst, 500 mM glucose, 10 mM MgCl 2 , in 50 mM KPi buffer (pH 7) without (top part of the figure) and with 0.1 mM NAD (bottom part of the figure).
  • the pH was adjusted manually with potassium hydroxide solution to the initial value.
  • FIG. 23 shows schematic representations of different vectors: a) vector pF(G)r7(A) and b) vector pFr3.
  • FIG. 24 shows a schematic representation of the two-step enzymatic reduction of dehydrocholic acid to 12-keto-ursodeoxycholic acid using two different whole-cell biocatalysts.
  • Reactions A and D are catalyzed by a 7 ⁇ -HSDH containing whole-cell biocatalyst
  • reactions B and C are catalyzed by a 3 ⁇ -HSDH containing whole-cell biocatalyst.
  • the intermediate 3,12-diketo-ursodeoxycholic acid must pass from the 7 ⁇ -HSDH containing whole-cell biocatalyst into the 3 ⁇ -HSDH containing whole-cell biocatalyst, and the intermediate 7,12-diketo-ursodeoxycholic acid must pass from the 3 ⁇ -HSDH containing whole-cell biocatalysts into the 7 ⁇ -HSDH containing whole-cell biocatalyst.
  • FIG. 25 shows the proportions of bile salts of biotransformation batches after 24 h when using 70 mM substrate (DHCA). The batches are shown when using different proportions of the two biocatalyst strains E. coli BL49 pF(G)r7(A) and E. coli BL49 pFr3.
  • FIG. 26 shows a time-resolved variation of biotransformation with the biocatalysts E. coli BL49 pF(G)r7(A) and E. coli BL49 pFr3 liter scale.
  • the batch contained 90 mM DHCA, 8.85 g/l BTM E. coli BL49 pF(G)r7(A) and 8.85 g/l BTM E. coli BL49 pFr3, 500 mM ammonium formate, 26% (v/v) glycerol, 50 mM MgCl 2 , in 50 mM KPi buffer (pH 6.5). Using pH adjustment, the pH was maintained with formic acid at pH 6.5 throughout the biotransformation.
  • a 7 ⁇ -hydroxysteroid dehydrogenase (7 ⁇ -HSDH) mutant which catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7-hydroxysteroid, wherein the mutant has a decreased substrate inhibition (especially for the 7-ketosteroid substrate) compared to the unmutated enzyme, such as in particular an enzyme comprising SEQ ID NO:2 and/or at least the sequence motif VMVGRRE according to position 36 to 42 thereof; and/or an altered cofactor usage (e.g. increased, altered specificity with respect to a cofactor (especially NADH or NADPH) or an extended dependence, i.e. usage of an additional cofactor not used previously).
  • a cofactor especially NADH or NADPH
  • an extended dependence i.e. usage of an additional cofactor not used previously.
  • K i value for the 7-ketosteroid substrate especially for dehydrocholic acid (DHCA)
  • DHCA dehydrocholic acid
  • a 7 ⁇ -hydroxysteroid dehydrogenase (7 ⁇ -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 has at least one mutation in the amino acid sequence according to SEQ ID NO:2 or an amino acid sequence derived therefrom with at least 60% sequence identity, e.g. 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.
  • a 7 ⁇ -hydroxysteroid dehydrogenase (7 ⁇ -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 has at least one mutation in the sequence motif VMVGRRE according to position 36 to 42 of SEQ ID NO:2 or in the corresponding sequence motif of an amino acid sequence derived therefrom with at least 60% sequence identity, e.g. 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.
  • suitable single mutants comprise: G39A, G39S, G39D, G39V, G39T, G39P, G39N, G39E, G39Q, G39H, G39R, G39K and G39W, and R40D, R40E, R40I, R40V, R40L, R40G, R40A.
  • Suitable double mutants comprise:
  • X 1 stands in particular for D or E and/or X 2 can be any amino acid, especially proteinogenic amino acid, such as in particular an amino acid with aliphatic side chain, for example (G39D, R40I), (G39D, R40L), (G39D, R40V); and the analogous double mutants with G39E instead of G39D; and double combinations of the above G39X 1 mutants or R40X 2 mutants with R41X 3 mutants, in which X 3 is any amino acid, especially an, especially natural, amino acid, that decreases substrate inhibition and/or that modifies cofactor usage or cofactor dependence, different from R, for example of the type (G39X 1 , R41X 3 ) or (R40X 2 , R41X 3 ), e.g.
  • the mutants of embodiments 1 to 6 can have, additionally or alternatively, especially additionally, at least one further substitution, for example 1, 2, 3 or 4 substitutions in the positions K44, R53, K61 and R64.
  • these residues can be replaced, independently of one another, with any amino acid, especially a proteinogenic amino acid, especially a substitution such that the resultant mutant has a decreased substrate inhibition (especially for the 7-ketosteroid substrate); and/or has an altered cofactor usage or cofactor dependence (e.g. increased, altered specificity with respect to a cofactor or an extended dependence, i.e. usage of an additional cofactor not used previously) as defined herein.
  • Expression cassette comprising a nucleic acid sequence according to embodiment 7 under the genetic control of at least one regulatory nucleic acid sequence, and optionally coding sequences for at least one (for example 1, 2 or 3) further enzyme, selected from hydroxysteroid dehydrogenases, especially 3 ⁇ -HSDH, and dehydrogenases suitable for cofactor regeneration, for example FDH, GDH, ADH, G-6-PDH, PDH.
  • the enzymes contained in an expression cassette can use different, but preferably the same pairs of cofactors, for example the pair of cofactors NAD + /NADH or NADP + /NADPH.
  • Vector comprising at least one expression cassette according to embodiment 8.
  • Recombinant microorganism bearing at least one nucleic acid sequence according to embodiment 7 or at least one expression cassette according to embodiment 8 or bearing at least one vector according to embodiment 9 and additionally, optionally, bearing the coding sequence for a 3 ⁇ -hydroxysteroid dehydrogenase (3 ⁇ -HSDH).
  • ketosteroid to be reduced is selected from
  • a process for enzymatic or microbial oxidation of 7 ⁇ -hydroxysteroids wherein the hydroxysteroid is reacted in the presence of a 7 ⁇ -HSDH mutant according to the definition in one of the embodiments 1 to 6 or in the presence of a microorganism expressing this mutant according to embodiment 10, and a resultant oxidation product is optionally isolated from the reaction mixture.
  • spent NADPH is regenerated by coupling with an NADPH-regenerating enzyme, wherein this is selected in particular from NADPH dehydrogenases, alcohol dehydrogenases (ADH), and NADPH regenerating formate dehydrogenases (FDH), and glucose dehydrogenase (GDH)-, glucose-6-phosphate dehydrogenase (G-6-PDH), or phosphite dehydrogenases (PtDH), wherein the NADPH-regenerating enzyme is optionally expressed by a recombinant microorganism; or wherein spent NADH is regenerated by coupling with an NADH-regenerating enzyme, wherein this is selected in particular from NADH dehydrogenases, NADH regenerating formate dehydrogenases (FDH), NADH regenerating alcohol dehydrogenases (ADH), NADH regenerating glucose-6-phosphate dehydrogenases (G6PDH), NADH regenerating phosphite dehydrogen
  • NADPH-regenerating enzyme is selected from natural or recombinant, isolated or enriched
  • NADPH-regenerating enzyme is selected from mutants of an NAD + -dependent formate dehydrogenase (FDH), which in particular catalyzes at least the enzymatic oxidation of formic acid to CO 2 , wherein the mutant accepts, compared to the unmutated enzyme, exclusively or additionally, especially additionally, NADP + as cofactor; or
  • FDH NAD + -dependent formate dehydrogenase
  • NADH-regenerating enzyme is selected from an NAD + -dependent FDH or an NAD + -dependent GDH, such as in particular an FDH from Mycobacterium vaccae N10, according to SEQ ID NO:36 and a GDH from Bacillus subtilis according to SEQ ID NO:48 (which regenerates NADPH and/or NADH) or a modified form thereof in each case functionally equivalent (with respect to cofactor regeneration) to the two stated enzymes.
  • NADP + -accepting FDH mutant has at least one mutation in the amino acid sequence of an FDH from Mycobacterium vaccae N10 according to SEQ ID NO:36 or an amino acid sequence derived therefrom with at least 60% sequence identity, e.g. 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.
  • NADP + -accepting mutant has at least one mutation in the sequence motif TDRHRL according to position 221 to 226 of SEQ ID NO:36 or in the corresponding sequence motif of an amino acid sequence derived therefrom with at least 60% sequence identity, e.g. 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.
  • Non-limiting examples of possibly suitable FDH mutants comprise mutations in the positions D222 and/or 8223 of SEQ ID NO:36.
  • NADP + -accepting mutant is selected from the single mutant D222G according to SEQ ID NO:36 (herein also more often designated as “D221G” mutant (with counting, starting from Ala in position 2 of SEQ ID NO:36 as first amino acid) or the corresponding single mutants of an amino acid sequence derived therefrom with at least 60% sequence identity, e.g. 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.
  • FDH mutants according to SEQ ID NO: 15, 19 and 35.
  • FDH enzymes are accessible starting from the wild-type enzymes that can be isolated e.g. from Candida boidinii or Pseudomonas sp, and insertion of at least one functional mutation corresponding to the above mutations for altering the cofactor specificity.
  • various studies in the prior art for improving various FDH properties such as chemical or thermal stability or catalytic activity. These are summarized e.g. in Tishkov et al., Biomolecular Engineering 23 (2006), 89-110.
  • single or multiple point mutations described there can be combined, for example for increasing enzyme stability, with the mutations described according to the invention for modified cofactor usage.
  • a nucleic acid sequence selected from nucleic acid sequences a) simultaneously coding for an FDH mutant according to one of the embodiments 22 to 24 and a 7 ⁇ -HSDH mutant according to one of the embodiments 1 to 6 and optionally a 3 ⁇ -HSDH; or b) simultaneously coding for an FDH mutant according to one of the embodiments 22 to 24 and an unmutated 7 ⁇ -HSDH and optionally a 3 ⁇ -HSDH; or c) coding for a fusion protein comprising an FDH mutant according to one of the embodiments 22 to 24 and a 7 ⁇ -HSDH mutant according to one of the embodiments 1 to 6 and optionally a 3 ⁇ -HSDH; or d) coding for a fusion protein comprising an FDH mutant according to one of the embodiments 22 to 24 and an unmutated 7 ⁇ -HSDH and optionally a 3 ⁇ -HSDH, e) simultaneously coding for FDH wild type and a 7 ⁇ -HSDH mutant according to one of the embodiments 1 to 6 and optionally a 3 ⁇ -HS
  • Expression cassette comprising a nucleic acid sequence according to embodiment 25 under the genetic control of at least one regulatory nucleic acid sequence, wherein the coding sequences, independently of one another, can be contained singly or multiply in the construct, for example in 2, 3, 4, 5, or 6 to 10 copies. Through selection of the appropriate copy number, optionally occurring activity differences in the individual expression products can be compensated.
  • a recombinant microorganism bearing at least one nucleic acid sequence according to embodiment 25 or at least one expression cassette according to embodiment 27 or bearing at least one vector according to embodiment 28.
  • a recombinant microorganism that is capable of simultaneous expression of 7 ⁇ -HSDH (wild type), an NADP + -accepting FDH mutant and/or the corresponding FDH wild type and optionally of 3 ⁇ -HSDH; or which is capable of simultaneous expression of 7 ⁇ -HSDH (wild type), a GDH described herein and optionally a 3 ⁇ -HSDH described herein.
  • a recombinant microorganism that is capable of simultaneous expression of a 7 ⁇ -HSDH mutant, an NADP + -accepting FDH mutant and/or the corresponding FDH wild type and optionally of 3 ⁇ -HSDH; or which is capable of simultaneous expression of a 7 ⁇ -HSDH mutant, a GDH described herein and optionally a 3 ⁇ -HSDH described herein.
  • the FDH mutant is a mutant according to the definition in one of the embodiments 20 to 24; and wherein the FDH wild type is an FDH from Mycobacterium vaccae N10 according to SEQ ID NO:36 or an FDH derived therefrom with at least 60% sequence identity, e.g. 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.
  • the FDH wild type is an FDH from Mycobacterium vaccae N10 according to SEQ ID NO:36 or an FDH derived therefrom with at least 60% sequence identity, e.g. 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.
  • 3 ⁇ -HSDH is an enzyme comprising an amino acid sequence according to SEQ ID NO: 6 or 8 or 22 or an amino acid sequence derived therefrom with at least 60% sequence identity, e.g. 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.
  • the recombinant microorganism according to one of the embodiments 29 to 33 bearing the coding sequences for 7 ⁇ -HSDH, FDH and 3 ⁇ -HSDH on one or more (different) expression constructs.
  • the invention therefore relates to recombinant microorganisms, which are modified (for example transformed) with a single-plasmid system, bearing the coding sequences for 7 ⁇ -HSDH or mutants thereof, FDH or mutants thereof and 3 ⁇ -HSDH or mutants thereof in one or more copies, for example in 2, 3, 4, 5, or 6 to 10 copies.
  • the invention therefore also relates to recombinant microorganisms, which are modified (for example transformed) with a single-plasmid system, bearing the coding sequences for 7 ⁇ -HSDH or mutants thereof, GDH or mutants thereof and 3 ⁇ -HSDH or mutants thereof in one or more copies, for example in 2, 3, 4, 5 or 6 to 10 copies.
  • the enzymes (7 ⁇ -HSDH, FDH and 3 ⁇ -HSDH or mutants thereof) can, however, also be contained on 2 or 3 separate, mutually compatible plasmids in one or more copies.
  • Suitable basis vectors for preparing single-plasmid systems and multicopy plasmids are known by a person skilled in the art. As examples we may mention, for single-plasmid system, e.g.
  • pET21a and for multicopy plasmids e.g. the duet vectors marketed by the company Novagen, such as pACYCDuet-1, pETDuet-1, pCDFDuet-1, pRSFDuet-1 and pCOLADuet-1.
  • Vectors of this kind, their compatibility with other vectors and microbial host strains are given e.g. in the “User Protocol TB340 Rev. E0305” of the company Novagen.
  • the optimal combination of enzymes for the generation of plasmid systems can be undertaken by a person skilled in the art without undue effort, taking into account the teaching of the present invention.
  • a person skilled in the art can, for example, depending on the cofactor specificity of the 7 ⁇ -HSDH enzyme used in each case, select the most suitable enzyme for cofactor regeneration, selected from the aforementioned dehydrogenases, especially FDH, GDH and the respective mutants thereof.
  • a first microorganism can be modified with a plasmid, which bears the coding sequence for an NADPH-dependent 7 ⁇ -HSDH mutant and an FDH mutant regenerating this cofactor according to the present invention, or which bears the coding sequence for an NADPH-dependent 7 ⁇ -HSDH mutant and NADPH-regenerating GDH, or the coding sequence for an NADH-dependent 7 ⁇ -HSDH and an NADH-regenerating FDH and/or GDH.
  • a second microorganism can, in contrast, be modified with a plasmid that bears the coding sequence for an NADH-dependent 3 ⁇ -HSDH and the coding sequence for an NADH-regenerating FDH wild type and/or for an NADH-regenerating GDH. Both microorganisms can then be used simultaneously for the biocatalytic reaction according to the invention.
  • the two biocatalysts can be genetically modified and optimized separately from one another. In particular it is possible to use different cofactor regeneration enzymes, which are either optimized for NADH regeneration or for NADPH regeneration.
  • the biocatalysts can be used in different proportions. This permits intervention in the individual reaction rates of the multienzyme process during biocatalysis, even after all biocatalysts have already been prepared.
  • R stands for alkyl, NR 1 R 2 , H, an alkali metal ion or N(R 3 ) 4 + , in which the residues R 3 may be identical or different and stand for H or alkyl, wherein a) optionally a cholic acid (CA) of formula (2)
  • DHCA is reduced in the presence of at least one 7 ⁇ -HSDH mutant (present as isolated enzyme or expressed by a corresponding recombinant microorganism) according to the definition in one of the embodiments 1 to 6 and in the presence of at least one 3 ⁇ -hydroxysteroid dehydrogenase (3 ⁇ -HSDH) (present as isolated enzyme or expressed by a corresponding recombinant microorganism) to the corresponding 12-keto-ursodeoxycholic acid (12-keto UDCA) of formula (5)
  • Process step b) can be configured differently. Either both enzymes (7 ⁇ -HSDH mutant and 3 ⁇ -HSDH) can be present simultaneously (e.g. one-pot reaction with both isolated enzymes or one or more corresponding recombinant microorganisms are present, which express both enzymes), or the partial reactions can take place in any order (first the 7 ⁇ -HSDH-mutant-catalyzed reduction and then the 3 ⁇ -HSDH-catalyzed reduction; or first the 3 ⁇ -HSDH-catalyzed reduction and then the 7 ⁇ -HSDH mutant-catalyzed reduction).
  • a process variant for the preparation of UDCA of formula (1) could therefore be for example as follows:
  • a) optionally a cholic acid (CA) of formula (2) is oxidized chemically; b) DHCA is reduced in the presence of at least one 7 ⁇ -HSDH mutant (present as isolated enzyme or expressed by a corresponding recombinant microorganism) according to the definition in one of the embodiments 1 to 6 to the 3,12-diketo-7 ⁇ -cholanic acid (3,12-diketo-7 ⁇ -CA) of formula (4)
  • 3,12-diketo-7 ⁇ -CA is reduced in the presence of at least one 3 ⁇ -hydroxysteroid dehydrogenase (3 ⁇ -HSDH) (present as isolated enzyme or expressed by a corresponding recombinant microorganism) to the corresponding 12-keto-ursodeoxycholic acid (12-keto UDCA) of formula (5)
  • steps b) and c) are carried out in the presence of one or more recombinant microorganisms described herein, such as at least one microorganism according to embodiment 10.
  • steps b) and/or c) are coupled to identical or different cofactor regeneration systems (present as isolated enzyme or expressed by a corresponding recombinant microorganism).
  • step b) is coupled to a cofactor regeneration system, in which NADPH is regenerated by an NADP + -accepting FDH mutant according to the definition in one of the embodiments 20 to 24 with consumption of formic acid or a salt thereof; or is coupled to a cofactor regeneration system, in which NADPH is regenerated by an ADH with consumption of isopropanol; or is coupled to a cofactor regeneration system in which NADPH is regenerated by a GDH with consumption of glucose; or is coupled to a cofactor regeneration system in which spent NADH is regenerated by an NADH regenerating GDH, ADH or FDH.
  • step c) is coupled to a cofactor regeneration step, in which NADPH is regenerated by an NADP + -accepting FDH mutant according to the definition in one of the embodiments 20 to 24 with consumption of formic acid or a salt thereof; or wherein step c) is coupled to a cofactor regeneration step in which NADH is regenerated by an NADH-regenerating FDH mutant according to the definition in one of the embodiments 20 to 24 or by the unmutated FDH with consumption of formic acid or a salt thereof or by an NADH-regenerating GDH with consumption of glucose.
  • R stands for alkyl, NR 1 R 2 , H, an alkali metal ion or N(R 3 ) 4 + , in which the residues R 3 may be identical or different and stand for H or alkyl, wherein a) optionally a cholic acid (CA) of formula (2)
  • DHCA is reduced in the presence of at least one 7 ⁇ -HSDH and in the presence of at least one 3 ⁇ -HSDH to the corresponding 12-keto-ursodeoxycholic acid (12-keto UDCA) of formula (5)
  • step b) take place microbially, i.e. in the presence of whole cells of one or more different recombinant microorganisms according to one of the embodiments 28 or 29 to 34, wherein the microorganism or microorganisms carry the enzymes necessary for the reaction and cofactor regeneration in a manner described in more detail herein, or else using at least one nucleic acid sequence according to embodiment 25.
  • DHCA can be reduced in the presence of at least one 78-HSDH or mutant thereof to 3,12-diketo-7 ⁇ -cholanic acid (3,12-diketo-7 ⁇ -CA) of formula (4)
  • 3,12-diketo-7 ⁇ -CA can be reduced in the presence of at least one 3 ⁇ -hydroxysteroid dehydrogenase (3 ⁇ -HSDH) or mutant thereof to the corresponding 12-keto-ursodeoxycholic acid (12-keto UDCA) of formula (5)
  • reaction sequence comprising the reduction of DHCA first with 3 ⁇ -HSDH and the subsequent reduction of the resultant reaction product with 7 ⁇ -HSDH, as well as both reaction sequences taking place simultaneously, on the basis of the simultaneous presence of both HSDHs.
  • a bioreactor for carrying out a process according to one of the embodiments 35 to 41 in particular containing at least one of the enzymes (7 ⁇ -HSDH, FDH, and/or 3 ⁇ -HSDH or mutants thereof; or 7 ⁇ -HSDH, GDH and/or 3 ⁇ -HSDH or mutants thereof) or a recombinant microorganism recombinantly expressing at least one of these enzymes, especially in immobilized form.
  • the present invention is not limited to the concrete embodiments described herein. Rather, a person skilled in the art will be enabled, through the teaching of the present invention, to provide further configurations of the invention without undue effort. He can, for example, also purposefully generate further enzyme mutants and screen and optimize these for the desired property profile (improved cofactor dependence and/or stability, reduced substrate inhibition); or isolate further suitable wild-type enzymes (7 ⁇ - and 3 ⁇ -HSDHs, FDHs, GDHs ADHs etc.) and use them according to the invention.
  • HSDHs used such as in particular 7 ⁇ -HSDH and 3 ⁇ -HSDH or mutants thereof
  • suitable dehydrogenases usable for cofactor regeneration GDH, FHD, ADH etc.
  • suitable dehydrogenases usable for cofactor regeneration GDH, FHD, ADH etc.
  • mutants thereof distribute the selected enzymes to one or more expression constructs or vectors and therefore if necessary produce one or more recombinant microorganisms, which then make an optimized whole-cell-based method of production possible.
  • 7 ⁇ -HSDH denotes a dehydrogenase enzyme, which catalyzes at least the stereospecific and/or regiospecific reduction of DHCA or 7,12-diketo-3 ⁇ -CA (7,12-diketo-LCA) to 3,12-diketo-7 ⁇ -CA or 12-keto-UDCA in particular with stoichiometric consumption of NADPH, and optionally the corresponding reverse reaction.
  • the enzyme can be a native or recombinantly produced enzyme.
  • the enzyme can basically be mixed with cellular, for example protein impurities, but preferably is in pure form. Suitable methods of detection are described for example in the experimental section given below or are known from the literature (e.g.
  • 3 ⁇ -HSDH denotes a dehydrogenase enzyme that catalyzes at least the stereospecific and/or regiospecific reduction of 3,12-diketo-7 ⁇ -CA or DHCA to 12-keto-UDCA or 7,12-diketo-3 ⁇ -CA (7,12-diketo-LCA), in particular with stoichiometric consumption of NADH and/or NADPH, and optionally the corresponding reverse reaction.
  • Suitable methods of detection are described for example in the experimental section given below or are known from the literature.
  • Suitable enzymes are obtainable e.g. from Comanomonas testosteroni (e.g. ATCC11996).
  • NADPH-dependent 3 ⁇ -HSDH is known for example from rodents and can also be used.
  • GDH denotes a dehydrogenase enzyme that catalyzes at least the oxidation of ⁇ -D-glucose to D-glucono-1,5-lactone with stoichiometric consumption of NAD + and/or NADP + and optionally the corresponding reverse reaction.
  • Suitable enzymes are obtainable e.g. from Bacillus subtilis or Bacillus megaterium . Enzymes with this activity are classified under EC number 1.1.1.47.
  • FDH denotes a dehydrogenase enzyme that catalyzes at least the oxidation of formic acid (or corresponding formate salts) to carbon dioxide with stoichiometric consumption of NAD + and/or NADP + , and optionally the corresponding reverse reaction.
  • Suitable methods of detection are for example described in the experimental section given below or are known from the literature.
  • Suitable enzymes are obtainable e.g. from Candida boidinii, Pseudomonas sp, or Mycobacterium vaccae . Enzymes with this activity are classified under EC number 1.2.1.2.
  • a “pure form” or a “pure” or “substantially pure” enzyme is to be understood according to the invention as an enzyme with a degree of purity above 80, preferably above 90, especially above 95, and quite particularly above 99 wt %, relative to the total protein content, determined by means of usual methods of detecting proteins, for example the biuret method or protein detection according to Lowry et al. (cf. description in R. K. Scopes, Protein Purification, Springer Verlag, New York, Heidelberg, Berlin (1982)).
  • a “redox equivalent” means a low-molecular organic compound usable as electron donor or electron acceptor, for example nicotinamide derivatives such as NAD + and NADH + or their reduced forms NADH and NADPH respectively.
  • “Redox equivalent” and “cofactor” are used as synonyms in the context of the present invention.
  • a “cofactor” in the sense of the invention can also be described as “redox-capable cofactor”, i.e. as a cofactor that can be present in a reduced and an oxidized form.
  • a “spent” cofactor is to be understood as the reduced or oxidized form of the cofactor, which in the course of a specified reduction or oxidation reaction of a substrate is transformed into the corresponding oxidized or reduced form. By regeneration, the oxidized or reduced cofactor form that is formed in the reaction is converted back to the reduced or oxidized starting form, so that it is available again for the reaction of the substrate.
  • an altered cofactor usage is to be understood in the context of the present invention as a qualitative or quantitative change compared to a reference.
  • an altered cofactor usage can be observed by undertaking amino acid sequence mutations. This change can then be determined compared to the unmutated starting enzyme. Moreover, the activity with respect to a particular cofactor can be increased or reduced by undertaking a mutation or can be prevented completely.
  • An altered cofactor usage also comprises however, changes such that instead of a specificity for an individual cofactor, now at least one further second cofactor, different from the first cofactor, is usable (i.e. there is an extended cofactor usage).
  • NADH cofactor NAD
  • NADPH cofactor NADP
  • NAD + /NADH dependence or “NADP + /NADPH dependence”, unless stated otherwise, are to be interpreted widely according to the invention. These terms comprise both “specific” dependences, i.e. exclusively dependence on NAD + /NADH or NADP/NADPH, as well as the dependence of the enzymes used according to the invention on both cofactors, i.e. dependence on NAD + /NADH and NADP + /NADPH.
  • NAD + /NADH-accepting or “NADP + /NADPH-accepting”.
  • NAD + /NADH-regenerating or “NADP + /NADPH-regenerating”, unless stated otherwise, are to be interpreted widely according to the invention. These terms comprise both “specific” i.e. exclusive capacity for regenerating spent cofactor NAD + /NADH or NADP + /NADPH, and the capacity for regenerating both cofactors, i.e. NAD + /NADH and NADP + /NADPH.
  • Proteinogenic amino acids comprise in particular (single-letter code): G, A, V, L, I, F, P, M, W, S, T, C, Y, N, Q, D, E, K, R and H.
  • “Immobilization” means, according to the invention, the covalent or noncovalent binding of a biocatalyst used according to the invention, for example a 7 ⁇ -HSDH on a solid, i.e. essentially insoluble in the surrounding liquid medium, carrier material. According to the invention, whole cells, such as the recombinant microorganisms used according to the invention, can correspondingly also be immobilized by means of such carriers.
  • a “substrate inhibition reduced in comparison with the unmutated enzyme” means that the substrate inhibition observed with the unmutated enzyme for a particular substrate is no longer observed, i.e. essentially is no longer measurable, or only occurs at higher substrate concentration, i.e. the K i value is increased.
  • “Cholic acid compound” means compounds according to the invention with the carbon skeleton structure, especially the steroid structure of cholic acid and the presence of keto and/or hydroxy or acyloxy groups in ring position 7 and optionally ring positions 3 and/or 12.
  • a compound of a special type for example a “cholic acid compound” or an “ursodeoxycholic acid compound” in particular also means derivatives of the underlying starting compound (for example cholic acid or ursodeoxycholic acid).
  • Said derivatives comprise “salts”, for example alkali metal salts such as lithium, sodium and potassium salts of the compounds; and ammonium salts, wherein an ammonium salt comprises the NH 4 + salt or those ammonium salts in which at least one hydrogen atom can be replaced with a C 1 -C 6 -alkyl residue.
  • Typical alkyl residues are, in particular, C 1 -C 4 -alkyl residues, such as methyl, ethyl, n- or i-propyl-, n-, sec- or tert-butyl, and n-pentyl and n-hexyl and the singly or multiply branched analogs thereof.
  • Alkyl esters of compounds according to the invention are, in particular, lower alkyl esters, for example C 1 -C 6 -alkyl esters.
  • lower alkyl esters for example C 1 -C 6 -alkyl esters.
  • 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, wherein the alkyl residues can optionally be further substituted independently of one another, for example with carboxyl, hydroxyl, halogen (such as F, Cl, Br, I), nitro and sulfonate groups.
  • “Acyl groups” are, in particular, nonaromatic groups with 2 to 4 carbon atoms, for example acetyl, propionyl and butyryl, and aromatic groups with an optionally substituted mononuclear aromatic ring, wherein suitable substituents are selected for example from hydroxyl, halogen (such as F, Cl, Br, I), nitro and C 1 -C 6 -alkyl groups, for example benzoyl or toluoyl.
  • suitable substituents are selected for example from hydroxyl, halogen (such as F, Cl, Br, I), nitro and C 1 -C 6 -alkyl groups, for example benzoyl or toluoyl.
  • hydroxysteroid compounds used or prepared according to the invention for example cholic acid, ursodeoxycholic acid, 12-keto-chenodeoxycholic acid, chenodeoxycholic acid and 7-keto-lithocholic acid, can be used in stereoisomerically pure form or in a mixture with other stereoisomers in the process according to the invention or obtained therefrom.
  • the compounds used or prepared are used or isolated in substantially stereoisomerically pure form.
  • the present invention is not limited to the concretely disclosed proteins or enzymes with 7 ⁇ -HSDH, FDH, GDH or 3 ⁇ -HSDH activity or mutants thereof, but rather also extends to functional equivalents thereof.
  • “Functional equivalents” or analogs of the concretely disclosed enzymes are, in the context of the present invention, polypeptides that are different from them, but still possess the desired biological activity, for example 7 ⁇ HSDH activity.
  • “functional equivalents” are to be understood as enzymes that have, in the test used for 7 ⁇ -HSDH, FDH, GDH or 3 ⁇ -HSDH activity, an activity that is higher or lower by at least 1%, e.g. at least 10% or 20%, e.g. at least 50% or 75% or 90% than that of a starting enzyme, comprising an amino acid sequence defined herein.
  • Functional equivalents are in addition preferably stable in the pH range from 4 to 11 and advantageously possess an optimal pH in a pH range from 6 to 10, such as in particular 8.5 to 9.5, and an optimal temperature in the range from 15° C. to 80° C. or 20° C. to 70° C., for example about 45 to 60° C. or about 50 to 55° C.
  • the 7 ⁇ -HSDH activity can be detected using various known tests. Without being restricted to this, we may mention a test using a reference substrate, e.g. CA or DHCA, under standardized conditions, as defined in the experimental section.
  • a reference substrate e.g. CA or DHCA
  • Tests for determining the FDH, GDH or 3 ⁇ -HSDH activity are also known per se.
  • “Functional equivalents” also means, in particular, “mutants”, which have in at least one sequence position of the aforementioned amino acid sequences an amino acid other than that concretely stated, but nevertheless possess one of the aforementioned biological activities. “Functional equivalents” therefore comprise the mutants obtainable by one or more, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, amino acid additions, substitutions, deletions and/or inversions, wherein the stated changes can occur in any sequence position, provided they lead to a mutant with the property profile according to the invention. Functional equivalence in particular also obtains when the patterns of reactivity between mutant and unaltered polypeptide coincide qualitatively, i.e. for example the same substrates are converted at different rates. Examples of suitable amino acid substitutions are presented in the following table:
  • Precursors are natural or synthetic precursors of the polypeptides with or without the desired biological activity.
  • salts means both salts of carboxyl groups and acid addition salts of amino groups of the protein molecules according to the invention.
  • Salts of carboxyl groups can be prepared in a manner known per se and comprise inorganic salts, for example sodium, calcium, ammonium, iron and zinc salts, and salts with organic bases, for example amines, such as triethanolamine, arginine, lysine, piperidine and the like.
  • Acid addition salts 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 also an object of the invention.
  • “Functional derivatives” of polypeptides according to the invention can also be prepared on functional amino acid side groups or on their N- or C-terminal end using known techniques.
  • Such derivatives comprise 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.
  • “Functional equivalents” naturally also comprise polypeptides that are obtainable from other organisms, and naturally occurring variants. For example, by sequence comparison, homologous sequence regions can be found and equivalent enzymes can be determined based on the concrete instructions of the invention.
  • “Functional equivalents” also comprise fragments, preferably individual domains or sequence motifs, of the polypeptides according to the invention, which for example have the desired biological function.
  • “Functional equivalents” are in addition fusion proteins that have one of the aforementioned polypeptide sequences or functional equivalents derived therefrom and at least one further, functionally different therefrom, heterologous sequence in functional N- or C-terminal linkage (i.e. without mutual substantial functional impairment of the fusion protein parts).
  • Nonlimiting examples of said heterologous sequences are e.g. signal peptides, histidine anchors or enzymes.
  • “Functional equivalents” that are also included according to the invention are homologs of the concretely disclosed proteins. These possess at least 60%, preferably at least 75%, especially at least 85%, for example 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, homology (or identity) to one of the concretely disclosed amino acid sequences, calculated according to the algorithm of Pearson and Lipman, Proc. Natl. Acad. Sci. (USA) 85(8), 1988, 2444-2448.
  • a percentage homology or identity of a homologous polypeptide according to the invention means, in particular, percentage identity of the amino acid residues relative to the total length of one of the amino acid sequences concretely described herein.
  • the percentage identity values can also be determined on the basis of BLAST alignments, the blastp (protein-protein BLAST) algorithm, or using the Clustal settings given below.
  • “functional equivalents” according to the invention comprise proteins of the type designated above in deglycosylated or glycosylated form and modified forms obtainable by altering the glycosylation pattern.
  • Homologs of the proteins or polypeptides according to the invention can be produced by mutagenesis, e.g. by point mutation, lengthening or shortening of the protein.
  • Homologs of the proteins according to the invention can be identified by screening combinatorial libraries of mutants, for example shortened mutants.
  • a variegated database of protein variants can be produced by combinatorial mutagenesis at the nucleic acid level, for example by enzymatic ligation of a mixture of synthetic oligonucleotides.
  • combinatorial mutagenesis at the nucleic acid level, for example by enzymatic ligation of a mixture of synthetic oligonucleotides.
  • degenerated set of genes makes it possible to provide all sequences in one mixture, which encode the desired set of potential protein sequences.
  • Methods of synthesis of degenerated oligonucleotides are known by a person skilled in the art (e.g. Narang, S. A. (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. 11: 477).
  • REM Recursive ensemble mutagenesis
  • the invention further comprises the use of the 7 ⁇ -HSDH wild type from Collinsella aerofaciens ATCC 25986, as described in the applicant's earlier international patent application PCT/EP2010/068576, which is expressly referred to hereby.
  • This 7 ⁇ -HSDH obtainable from Collinsella aerofaciens DSM 3979 is in particular characterized by at least one other of the following properties, for example 2, 3, 4, 5, 6 or 7 or all such properties:
  • molecular weight (SDS-gel electrophoresis): about 28-32 kDa, especially about 29 to 31 kDa or about 30 kDa;
  • molecular weight (gel filtration, in nondenaturing conditions, such as in particular without SDS): about 53 to 60 kDa, especially about 55 to 57 kDa, such as 56.1 kDa. This proves the dimeric nature of the 7 ⁇ -HSDH from Collinsella aerofaciens DSM 3979;
  • this 7 ⁇ -HSDH shows 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).
  • a 7 ⁇ -HSDH of this kind or a functional equivalent derived therefrom is moreover characterized by
  • Said 7 ⁇ -HSDH has in particular an amino acid sequence according to SEQ ID NO:2 (accession No.: ZP — 01773061) or a sequence derived therefrom with a degree of identity of at least 60%, e.g. at least 65, 70, 75, 80, 85, or 90, e.g.
  • the invention also relates to nucleic acid sequences that code for an enzyme with 7 ⁇ -HSDH, FDH, GDH and/or 3 ⁇ -HSDH activity and the mutants thereof.
  • the present invention also relates to nucleic acids with a specified degree of identity to the concrete sequences described herein.
  • Identity between two nucleic acids means the identity of the nucleotides over the total nucleic acid length in each case, especially the identity that is calculated by comparison by means of the Vector NTI Suite 7.1 software of the company Informax (USA) employing the Clustal method (Higgins D G, Sharp P M. Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl. Biosci. 1989 April; 5(2): 151-1) setting the following parameters:
  • the identity can also be determined according to Chema, 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 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 Gonnet Protein/DNA ENDGAP ⁇ 1 Protein/DNA GAPDIST 4
  • nucleic acid sequences mentioned herein can be produced in a manner known per se by chemical synthesis from the nucleotide building blocks, for example by fragment condensation of individual overlapping, complementary nucleic acid building blocks of the double helix.
  • the chemical synthesis of oligonucleotides can be carried out for example in a known manner by the phosphoroamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897).
  • the invention also relates to nucleic acid sequences (single- and double-stranded DNA and RNA sequences, for example cDNA and mRNA) coding for one of the above polypeptides and functional equivalents thereof, which are accessible for example using artificial nucleotide analogs.
  • the invention relates both to isolated nucleic acid molecules, which code for polypeptides or proteins according to the invention or biologically active segments thereof, and to nucleic acid fragments that can be used e.g. as hybridization probes or primers for identification or amplification of coding nucleic acids according to the invention.
  • nucleic acid molecules according to the invention can moreover 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 concretely described nucleotide sequences, or a segment thereof.
  • nucleotide sequences according to the invention make it possible to produce probes and primers that can be used for identification and/or cloning of homologous sequences in other cell types and organisms.
  • These probes or primers usually comprise a nucleotide sequence region, which hybridizes under “stringent” conditions (see below) to at least about 12, preferably at least about 25, for example about 40, 50 or 75 successive nucleotides of a sense strand of a nucleic acid sequence according to the invention or a corresponding antisense strand.
  • nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid and can moreover be essentially free from other cellular material or culture medium, when it is produced by recombinant techniques, or free from chemical precursors or other chemicals, when it is synthesized chemically.
  • a nucleic acid molecule according to the invention can be isolated using standard techniques of molecular biology and the sequence information provided according to the invention.
  • cDNA can be isolated from a suitable cDNA library, using one of the concretely disclosed complete sequences or a segment thereof as hybridization probe and standard hybridization techniques (as described for example in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
  • a nucleic acid molecule comprising one of the disclosed sequences or a segment thereof, can be isolated by polymerase chain reaction, using the oligonucleotide primers that are prepared on the basis of this sequence.
  • the nucleic acid thus amplified can be cloned into a suitable vector and can be characterized by DNA sequence analysis.
  • the oligonucleotides according to the invention can also be produced by standard synthesis techniques, e.g. with an automatic DNA synthesizer.
  • Nucleic acid sequences according to the invention or derivatives thereof, homologs or parts of these sequences can be isolated for example with usual hybridization methods or PCR technology from other bacteria, e.g. via genomic or cDNA libraries. These DNA sequences hybridize under standard conditions to the sequences according to the invention.
  • Hybridize means the capacity of a polynucleotide or oligonucleotide for binding to an almost complementary sequence under standard conditions, whereas under these conditions nonspecific bindings do not occur between noncomplementary partners.
  • sequences can be complementary to 90-100%.
  • the property of complementary sequences of being able to bind specifically to one another is utilized for example in Northern or Southern blotting or in primer binding in PCR or RT-PCR.
  • short oligonucleotides of the conserved regions are used for hybridization. It is also possible, however, to use longer fragments of the nucleic acids according to the invention or the complete sequences for hybridization. These standard conditions vary depending on the nucleic acid used (oligonucleotide, longer fragment or complete sequence) or depending on which type of nucleic acid, DNA or RNA, is used for the hybridization. Thus, for example, the melting points for DNA:DNA hybrids are approx. 10° C. lower than those of DNA:RNA hybrids of the same length.
  • the hybridization conditions for DNA:DNA hybrids are 0.1 ⁇ SSC and temperatures between about 20° C. and 45° C., preferably between about 30° C. and 45° C.
  • the hybridization conditions are advantageously 0.1 ⁇ SSC and temperatures between about 30° C. and 55° C., preferably between about 45° C. and 55° C.
  • Hybridization can in particular take place under stringent conditions. These hybridization conditions are described for example in Sambrook, J., Fritsch, E. F., Maniatis, T., in: Molecular Cloning (A Laboratory Manual), 2nd edition, Cold Spring Harbor Laboratory Press, 1989, pages 9.31-9.57 or in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • “Stringent” hybridization conditions are understood in particular as: incubation at 42° C. overnight in a solution consisting of 50% formamide, 5 ⁇ SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5 ⁇ Denhardt solution, 10% dextran sulfate and 20 g/ml denatured, sheared salmon sperm DNA, followed by a filter washing step with 0.1 ⁇ SSC at 65° C.
  • the invention also relates to derivatives of the concretely disclosed or derivable nucleic acid sequences.
  • nucleic acid sequences according to the invention can be derived e.g. from SEQ ID NO:1, 5, 7, 14, 19, 34 or 47 and differ from them by addition, substitution, insertion or deletion of individual or several nucleotides, but furthermore code for polypeptides with the desired property profile.
  • the invention also covers those nucleic acid sequences that comprise so-called silent mutations or are altered corresponding to the codon usage of a special original or host organism, compared to a concretely stated sequence, as well as naturally occurring variants, for example splice variants or allele variants, thereof.
  • the invention also relates to sequences obtainable by conservative nucleotide substitutions (i.e. the amino acid in question is replaced with an amino acid of the same charge, size, polarity and/or solubility).
  • the invention also relates to the molecules derived by sequence polymorphisms from the concretely disclosed nucleic acids. These genetic polymorphisms can exist between individuals within a population owing to natural variation. These natural variations usually bring about a variance from 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:1, 5, 7, 14, 19, 34 or 47 mean for example allele variants that have at least 60% homology at the derived amino acid level, preferably at least 80% homology, quite especially preferably at least 90% homology over the total sequence region (regarding homology at the amino acid level, reference may be made to the above information for the polypeptides).
  • the homologies can advantageously be higher on partial regions of the sequences.
  • derivatives are also to be understood as homologs of the nucleic acid sequences according to the invention, especially of SEQ ID NO:1, 5, 7, 14, 19, 34 or 47, for example fungal or bacterial homologs, shortened sequences, single-stranded DNA or RNA of the coding and noncoding DNA sequence.
  • homologs to SEQ ID NO:1, 5, 7, 14, 19, 34 or 47 possess, at DNA level, a homology of at least 40%, preferably of at least 60%, especially preferably of at least 70%, quite especially preferably of at least 80% over the whole DNA region given in SEQ ID NO:1, 5, 7, 14, 19, 34 or 47.
  • derivatives are to be understood for example as fusions with promoters.
  • the promoters that precede the stated nucleotide sequences can be altered by at least one nucleotide exchange, at least one insertion, inversion and/or deletion, but without the functionality or effectiveness of the promoters being impaired.
  • the effectiveness of the promoters can be increased by altering their sequence or they can be exchanged completely with more effective promoters even of organisms of a different species.
  • a person skilled in the art can insert completely random or even more targeted mutations in genes or also noncoding nucleic acid regions (which are for example important for the regulation of expression) and then prepare gene banks.
  • the methods of molecular biology required for this are known by a person skilled in the art and for example are described in Sambrook and Russell, Molecular Cloning. 3rd edition, Cold Spring Harbor Laboratory Press 2001.
  • error-prone PCR the error-prone polymerase chain reaction
  • DNA polymerases in which nucleotide sequences are mutated by incorrectly functioning DNA polymerases
  • directed evolution (described inter alia in Reetz M T and Jaeger K-E (1999), Topics Curr Chem 200: 31; Zhao H, Moore J C, Volkov A A, Arnold F H (1999), Methods for optimizing industrial enzymes by directed evolution, In: Demain A L, Davies J E (Publ.) Manual of industrial microbiology and biotechnology. American Society for Microbiology), a person skilled in the art can produce functional mutants in a targeted manner and on a large scale.
  • a first step firstly gene banks of the respective proteins are produced, for example using the methods given above.
  • the gene banks are expressed in a suitable manner, for example by bacteria or by phage-display systems.
  • the relevant genes of host organisms that express functional mutants with properties that largely correspond to the desired properties can be submitted to another round of mutation.
  • the steps of mutation and of selection or screening can be repeated iteratively until the functional mutants present have the desired properties to a sufficient degree.
  • a limited number of mutations for example 1 to 5 mutations, can be performed in steps and assessed and selected for their influence on the relevant enzyme property.
  • the selected mutant can then be submitted to another mutation step in the same way. As a result, the number of individual mutants to be investigated can be reduced significantly.
  • results according to the invention provide important information regarding structure and sequence of the enzymes in question, which is necessary for targeted generation of further enzymes with desired modified properties.
  • so-called “hot spots” can be defined, i.e. sequence segments that are potentially suitable for modifying an enzyme property by introducing targeted mutations.
  • the invention further relates to expression constructs containing, 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.
  • “Expression unit” means, according to the invention, a nucleic acid with expression activity, which comprises a promoter, as defined herein, and, after functional linking with a nucleic acid to be expressed or a gene, regulates the expression, i.e. the transcription and the translation of this nucleic acid or of this gene. Therefore the term “regulatory nucleic acid sequence” is also used in this context. In addition to the promoter, other regulatory elements, for example enhancers, can be present.
  • “Expression cassette” or “expression construct” means, according to the invention, an expression unit that is functionally linked to the nucleic acid to be expressed or the gene to be expressed.
  • an expression cassette therefore comprises not only nucleic acid sequences that regulate transcription and translation, but also the nucleic acid sequences that should be expressed as protein as a result of the transcription and translation.
  • expression or “overexpression” describe, in the context of the invention, the production or increase in the intracellular activity of one or more enzymes in a microorganism, which are encoded by the corresponding DNA.
  • a gene can be inserted in an organism, a gene that is present can be replaced with another gene, the copy number of the gene or genes can be increased, a strong promoter can be used or a gene can be used that codes for a corresponding enzyme with a high activity, and these measures can optionally be combined.
  • said constructs according to the invention comprise a promoter 5′-upstream of the respective coding sequence and 3′-downstream a terminator sequence and optionally further usual regulatory elements, in each case operatively linked with the coding sequence.
  • Promoter a “nucleic acid with promoter activity” or a “promoter sequence” mean, according to the invention, a nucleic acid, which in functional linkage with a nucleic acid to be transcribed, regulates the transcription of said nucleic acid.
  • “Functional” or “operational” linkage means in this context for example the sequential arrangement of one of the nucleic acids with promoter activity and a nucleic acid sequence to be transcribed and optionally further regulatory elements, for example nucleic acid sequences that ensure the transcription of nucleic acids, and for example a terminator, in such a way 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 linkage in the chemical sense. Genetic control sequences, for example enhancer sequences, can exert their function on the target sequence even from more remote positions or even from other DNA molecules. Arrangements are preferred in which the nucleic acid sequence to be transcribed is positioned behind the promoter sequence (i.e. at the 3′-end), so that the two sequences are linked together covalently.
  • the distance between the promoter sequence and the nucleic acid sequence that is to undergo transgene expression can be less than 200 base pairs, or less than 100 base pairs or less than 50 base pairs.
  • regulatory elements In addition to promoters and terminators, examples of other regulatory elements that may be mentioned are targeting sequences, enhancers, polyadenylation signals, selectable markers, amplification signals, replication origins and the like. Suitable regulatory sequences are described for example in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).
  • Nucleic acid constructs according to the invention comprise, in particular, sequence SEQ ID NO:1, 5, 7, 14, 19, 34 or 47 or derivatives and homologs thereof, and the nucleic acid sequences that can be derived therefrom, which can advantageously be linked operationally or functionally with one or more regulatory signals for controlling, e.g. increasing, gene expression.
  • the natural regulation of these sequences can still be present before the actual structural genes and optionally can have been genetically modified, so that the natural regulation is switched off and expression of the genes is increased.
  • the nucleic acid construct can also be of simpler construction, i.e. no additional regulatory signals have been inserted before the coding sequence and the natural promoter with its regulation has not been removed. Instead, the natural regulatory sequence is mutated so that regulation no longer occurs and gene expression is increased.
  • a preferred nucleic acid construct advantageously also contains one or more of the aforementioned “enhancer” sequences, functionally linked to the promoter, which make increased expression of the nucleic acid sequence possible. Additional advantageous sequences can also be inserted at the 3′-end of the DNA sequences, such as further regulatory elements or terminators.
  • the construct can contain one or more copies of the nucleic acids according to the invention.
  • the construct can also contain further markers, such as antibiotic resistances or auxotrophic complementation genes, optionally for selection of the construct.
  • suitable regulatory sequences are contained in promoters such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacI q- , T7, T5, T3, gal, trc, ara, rhaP (rhaP BAD )SP6, lambda-P R or in the lambda-P L promoter, which advantageously find application in Gram-negative bacteria.
  • Further advantageous regulatory sequences are contained for example in the Gram-positive promoters amy and SPO2, in the yeast or fungus promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH. Artificial promoters can also be used for regulation.
  • the nucleic acid construct is advantageously inserted into a vector, for example a plasmid or a phage, which makes optimal expression of the genes in the host possible.
  • a vector for example a plasmid or a phage
  • vectors are also to be understood as all other vectors known by a person skilled in the art, e.g. viruses, such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids, and linear or circular DNA.
  • viruses such as SV40, CMV, baculovirus and adenovirus
  • transposons e.g. viruses, such as SV40, CMV, baculovirus and adenovirus
  • transposons e.g. viruses, such as SV40, CMV, baculovirus and adenovirus
  • transposons e.g. viruses, such as SV40, CMV, baculovirus and adenovirus
  • Suitable plasmids are for example pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III 113 -B1, ⁇ gt11 or pBdCl in E.
  • coli coli , pIJ101, pIJ364, pIJ702 or pIJ361 in Streptomyces , pUB110, pC194 or pBD214 in Bacillus , pSA77 or pAJ667 in Corynebacterium , pALS1, pIL2 or pBB116 in fungi, 2alphaM, pAG-1, YEp6, YEp13 or pEMBLYe23 in yeasts or pLGV23, pGHlac + , pBIN19, pAK2004 or pDH51 in plants.
  • the aforementioned plasmids represent a small selection of the possible plasmids.
  • the vector containing the nucleic acid construct according to the invention or the nucleic acid according to the invention can also advantageously be inserted in the form of a linear DNA into the microorganisms and can be integrated by heterologous or homologous recombination into the genome of the host organism.
  • This linear DNA can consist of a linearized vector such as a plasmid or only of the nucleic acid construct or the nucleic acid according to the invention.
  • An expression cassette according to the invention is prepared by fusion of a suitable promoter with a suitable coding nucleotide sequence and a terminator or polyadenylation signal.
  • usual recombination and cloning techniques are used, such as are described for example in T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) and in T. J. Silhavy, M. L. Berman and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and in 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 inserted into a host-specific vector, which makes optimal expression of the genes in the host possible.
  • Vectors are well known by a person skilled in the art and can be found for example in “Cloning Vectors” (Pouwels P. H. et al., Publ., Elsevier, Amsterdam-New York-Oxford, 1985).
  • microorganism means the starting (wild-type) microorganism or a genetically modified, recombinant microorganism, or both.
  • recombinant microorganisms can be produced, which for example have been transformed with at least one vector according to the invention and can be used for producing the polypeptides according to the invention.
  • the recombinant constructs according to the invention described above are introduced into a suitable host system and expressed.
  • common cloning and transfection methods known by a person skilled in the art are used, for example coprecipitation, protoplast fusion, electroporation, retroviral transfection and the like, to bring about expression of the stated nucleic acids in the respective expression system. Suitable systems are described for example in Current Protocols in Molecular Biology, F.
  • prokaryotic or eukaryotic organisms may come into consideration as recombinant host organisms for the nucleic acid according to the invention or the nucleic acid construct.
  • microorganisms such as bacteria, fungi or yeasts are used as host organisms.
  • Gram-positive or Gram-negative bacteria are used, preferably bacteria of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae or Nocardiaceae, especially preferably bacteria of the genera Escherichia, Pseudomonas, Streptomyces, Nocardia, Burkholderia, Salmonella, Agrobacterium, Clostridium or Rhodococcus .
  • the genus and species Escherichia coli is quite especially preferred.
  • Further advantageous bacteria can be found, moreover, in the group of alpha-proteobacteria, beta-proteobacteria or gamma-proteobacteria.
  • the host organism or the 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 7 ⁇ -HSDH activity according to the above definition.
  • the organisms used in the process according to the invention are grown or cultured in a manner known by a person skilled in the art, depending on the host organism.
  • Microorganisms are as a rule grown in a liquid medium, which contains a carbon source generally in the form of sugars, a nitrogen source generally 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. and 60° C. with oxygen aeration.
  • the pH of the liquid nutrient medium can be maintained at a fixed value, i.e. during growing it may or may not be regulated.
  • Culture can be batchwise, semi-batchwise or continuous. Nutrients can be supplied at the start of fermentation or can be replenished semi-continuously or continuously.
  • Step 1 Chemical Reaction from CA to DHCA
  • hydroxy groups of CA are oxidized with chromic acid or chromates in acidic solution (e.g. H 2 SO 4 ) to carbonyl groups in a manner known per se by the classical chemical route.
  • acidic solution e.g. H 2 SO 4
  • Step 2 Enzymatic or Microbial Conversion of DHCA to 12-keto-UDCA
  • DHCA is reduced by 3 ⁇ -HSDH and 7 ⁇ -HSDH or mutants thereof specifically to 12-keto-UDCA in the presence of NADPH or NADH.
  • the cofactor NADPH or NADH can be regenerated by an ADH or FDH or GDH or mutants thereof from isopropanol or sodium formate or glucose.
  • recombinant microorganisms that express the necessary enzyme activity/activities can be cultured in the presence of the substrate to be converted (DHCA) anaerobically or aerobically in suitable liquid media.
  • suitable cultivation conditions are known per se by a person skilled in the art. They comprise reactions in the pH range of for example 5 to 10 or 6 to 9, at temperatures in the range from 10 to 60 or 15 to 45 or 25 to 40 or 37° C.
  • Suitable media comprise for example the LB and TB media described below.
  • the reaction can take place for example batchwise or continuously or in other usual process variants (as described above).
  • the reaction time can for example range from minutes to several hours or days, and can be e.g. 1 h to 48 h.
  • Step 3 Chemical Conversion of 12-keto-UDCA to UDCA
  • the 12-carbonyl group of 12-keto-UDCA is removed by means of Wolff-Kishner reduction in a manner known per se, with formation of UDCA from 12-keto-UDCA.
  • the carbonyl group is reacted with hydrazine to hydrazone.
  • the hydrazone is heated in the presence of a base (e.g. KOH) to 200° C., with cleavage of nitrogen and formation of UDCA.
  • a base e.g. KOH
  • the invention further relates to processes for recombinant production of polypeptides according to the invention or functional, biologically active fragments thereof, wherein a polypeptide-producing microorganism is cultured, optionally expression of the polypeptides is induced and the latter are isolated from the culture.
  • the polypeptides can also be produced on an industrial scale in this way, if this is desirable.
  • the microorganisms produced according to the invention can be cultured continuously or discontinuously in the batch method (batch culture) or in the fed batch or repeated fed batch method.
  • a summary of known cultivation methods can be found in Chmiel's textbook (Bioproze ⁇ technik 1. Lecture in die Biovonstechnik [Bioprocess technology 1. Introduction to bioprocess engineering] (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren and periphere saw [Bioreactors and peripheral equipment]) (Vieweg Verlag, Brunswick/Wiesbaden, 1994)).
  • the culture medium to be used must suitably fulfill the requirements of the respective strains. Descriptions of culture media for various microorganisms are given in the manual “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981).
  • These media usable 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. Very good carbon sources are for example glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars 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 various carbon sources.
  • oils and fats such as soybean oil, sunflower oil, peanut oil and coconut oil, 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 that contain these compounds.
  • nitrogen sources comprise 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 flour, soybean protein, yeast extract, meat extract and others.
  • the nitrogen sources can be used individually or as a mixture.
  • Inorganic salt compounds that can be contained in the media comprise the chloride, phosphorus or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
  • the sulfur source used can be inorganic sulfur compounds such as sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides but also organic sulfur compounds, such as mercaptans and thiols.
  • the phosphorus source used can be phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts.
  • Chelating agents can be added to the medium in order to keep the metal ions in solution.
  • suitable chelating agents comprise 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 components of the media, such as yeast extract, molasses, corn-steep liquor and the like.
  • suitable precursors can be added to the culture medium. The exact composition of compounds in the medium is strongly dependent on the particular experiment and is decided individually for each specific case. Information on media optimization can be found in the textbook “Applied Microbiol. Physiology, A Practical Approach” (Publ. P. M. Rhodes, P. F. Stanbury, IRL Press (1997) p. 53-73, ISBN 0 19 963577 3).
  • Growth media can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.
  • All components of the media are sterilized, either with heat (20 min at 1.5 bar and 121° C.) or by sterile filtration.
  • the components can either be sterilized together or separately if necessary.
  • All components of the media can be present at the start of culture or can optionally be added continuously or batchwise.
  • the culture temperature is normally between 15° C. and 45° C., preferably at 25° C. to 40° C. and can be kept constant or varied during the experiment.
  • the pH of the medium should be in the range from 5 to 8.5, preferably around 7.0.
  • the culture pH can be controlled during culture by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acid compounds such as phosphoric acid or sulfuric acid.
  • antifoaming agents such as fatty acid polyglycol esters.
  • suitable selectively acting substances such as antibiotics, can be added to the medium.
  • oxygen or oxygen-containing gas mixtures such as ambient air, are fed into the culture.
  • the culture temperature is normally at 20° C. to 45° C. Culture is continued until a maximum of the desired product has formed. This target is normally reached within 10 hours to 160 hours.
  • the fermentation broth is then processed further.
  • the biomass is removed from the fermentation broth completely or partially by separation techniques, such as centrifugation, filtration, decanting or a combination of these methods or can be left in it completely.
  • the cells can also be disrupted and the product can be obtained from the lysate by known methods of protein isolation.
  • the cells can optionally be disrupted with high-frequency ultrasound, by high pressure, for example in a French press, by osmolysis, by the action of detergents, lytic enzymes or organic solvents, using homogenizers or by a combination of several of the methods listed.
  • the polypeptides can be purified by known chromatographic methods, such as molecular sieve chromatography (gel filtration), such as Q-sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, and with other usual methods such as ultrafiltration, crystallization, salting-out, dialysis and native gel electrophoresis. Suitable methods are described for example in Cooper, F. G., Biochemische Anlagenmann, Berlin, New York or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.
  • vector systems or oligonucleotides that lengthen the cDNA with defined nucleotide sequences and therefore code for modified polypeptides or fusion proteins, which for example serve for easier purification.
  • Suitable modifications of this kind are for example so-called “tags” functioning as anchors, for example the modification known as hexa-histidine anchors, or epitopes that can be recognized as antigens by antibodies (described for example in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor (N.Y.) Press).
  • These anchors can serve for attaching the proteins to a solid carrier, for example a polymer matrix, which can for example be packed in a chromatography column, or can be used on a microtiter plate or on some other support.
  • these anchors can also be used for recognizing the proteins.
  • markers such as fluorescent dyes, enzyme markers, which form a detectable reaction product after reaction with a substrate, or radioactive markers, can be used, alone or in combination with the anchors for derivatization of the proteins.
  • the enzymes according to the invention can be used free or immobilized.
  • An immobilized enzyme is to be understood as an enzyme that is fixed to an inert support. Suitable support materials and the enzymes immobilized thereon are known from EP-A-1149849, EP-A-1 069 183 and DE-OS 100193773 and from the literature references cited therein. Regarding this, full reference is made to the disclosure of these documents.
  • Suitable support materials include for example clays, clay minerals, such as kaolinite, diatomaceous earth, perlite, silicon dioxide, aluminum oxide, sodium carbonate, calcium carbonate, cellulose powder, anion exchanger materials, synthetic polymers, such as polystyrene, acrylic resins, phenol-formaldehyde resins, polyurethanes and polyolefins, such as polyethylene and polypropylene.
  • the support materials are usually used in a finely-divided, particulate form, with porous forms being preferred.
  • the particle size of the support material is usually not more than 5 mm, especially not more than 2 mm (particle-size distribution curve).
  • a free or immobilized form can be selected.
  • Support materials are for example Ca-alginate, and carrageenan.
  • Enzymes as well as cells can also be crosslinked directly with glutaraldehyde (crosslinking to CLEAs). Corresponding and further methods of immobilization are described for example in J. Lalonde and A. Margolin “Immobilization of Enzymes” and in K. Drauz and H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol. III, 991-1032, Wiley-VCH, Weinheim.
  • the cloning steps carried out in the context of the present invention for example restriction cleavage, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids onto nitrocellulose and nylon membranes, linkage of DNA fragments, transformation of microorganisms, culturing of microorganisms, multiplication of phages and sequence analysis of recombinant DNA are carried out as described in Sambrook et al. (1989) op. cit.
  • the genomic DNA of Collinsella aerofaciens DSM 3979 was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ).
  • DSMZ German Collection of Microorganisms and Cell Cultures
  • UDCA and 7-keto-LCA are starting compounds that are known per se and are described in the literature. All other chemicals were obtained from Sigma-Aldrich and Fluka (Germany). All restriction endonucleases, T4 DNA ligase, Taq DNA polymerase, Phusion DNA polymerase and isopropyl- ⁇ -D-1-thiogalactopyranoside (IPTG) were obtained from Fermentas (Germany).
  • TB medium containing tryptone 12 g, yeast extract 24 g, 4 mL glycerol, 10% TB buffer (11.55 g KH 2 PO 4 , 62.7 g K 2 HPO 4 , H 2 O to 500 mL) per liter of medium
  • the vectors pET21a(+) and pET22b(+) each possess a multiple cloning site (MCS), in each case under the control of a T7-promoter with downstream lac-operator and the subsequent ribosomal binding site (rbs). In the C-terminal region of the expression domain there is in each case a T7-terminator.
  • Both plasmids have a ColE1-replicon (pBR322-replicon), an ampicillin resistance gene (bia), an f1-origin and a gene coding for the lac-inhibitor (lacI).
  • the pET21a(+)-plasmid has a T7.Tag in the N-terminal region of the MCS and an optional His.Tag C-terminally of the MCS.
  • the pET22b(+)-plasmid has, in the N-terminal region of the MCS, a pelB signal sequence and a His.Tag C-terminally of the MCS.
  • the pCOLADuet-1 vector has two MCSs, each of which is under the control of a T7-promoter with downstream lac-operator and subsequent ribosomal binding site (rbs). C-terminally of the two MCSs there is a T7-terminator. Moreover, this vector has a gene that codes for the lac-inhibitor and a COLA-replicon (ColA-replicon).
  • a modified variant of the commercially available pCOLADuet-1 vector was used in the present work.
  • this modified plasmid variant (designated pCOLA(mod); cf. FIG. 3 c )) the kanamycin resistance gene of the original vector was replaced with a chloramphenicol resistance gene.
  • an NcoI restriction site was removed from the chloramphenicol resistance gene by site-directed point mutagenesis.
  • His.Tag In the N-terminal region of the first MCS there is a His.Tag, whereas C-terminally of the second MCS there is an S.Tag.
  • the ColE1-replicons of the pET-plasmids and the COLA-replicon of the pCOLA-plasmid are mutually compatible. This permits simultaneous stable insertion of a pET-plasmid and of a pCOLA-plasmid in Escherichia coli . In this way, combinations of various genes can be cloned into Escherichia coli , without their being located on the same operon. Owing to the different copy numbers of pET-vectors ( ⁇ 40) and pCOLA-vectors (20-40), it is moreover possible to influence the expression level of cotransformed genes.
  • pET22b(+) 7 ⁇ -HSDH a pET22b(+) vector into which the 7 ⁇ -HSDH from Collinsella aerofaciens ATCC 25986 had been cloned via the Nde I and Hind III cleavage sites in the usual way.
  • pET22b(+) 3 ⁇ -HSDH a pET22b(+) vector into which the 3 ⁇ -HSDH from Comamonas testosteroni had been cloned via the Nde I and EcoR I cleavage sites in the usual way (Oppermann et al., J Biochem, 1996, 241(3): 744-749).
  • pET21a(+) FDH D221G (cf. FIG. 4 a ): a pET21a(+) vector into which the formate dehydrogenase from Mycobacterium vaccae N10 had been cloned via the Nde I and EcoR I cleavage sites.
  • the aspartate residue (D) at position 221 (without taking into account methionine in position 1) or position 222 (counting from methionine in position 1; cf. SEQ ID NO: 15, 19, 35) of formate dehydrogenase was replaced with a glycine residue (cf. production example 4, below).
  • Formate dehydrogenase carries, at position 1202 of the nucleotide sequence, a single base deletion, which leads to exchange of the last amino acid valine for an alanine. Simultaneously, this base deletion leads to switching-off of the stop codon and to activation of the His.Tag that was originally outside of the reading frame (cf. SEQ ID NO: 34 and 35).
  • pET21a(+) 7 ⁇ -HSDH a pET21a(+) vector, into which the 7 ⁇ -HSDH from Collinsella aerofaciens ATCC 25986 had been cloned via the Nde I and Xho I cleavage sites in the usual way
  • the Escherichia coli strain DH5 ⁇ (Novagen, Madison, Wis., USA) was multiplied at 37° C. in LB medium containing suitable antibiotics.
  • the reaction mixture contains a total volume of 1 ml:
  • the reaction mixture contains a total volume of 1 ml
  • the samples were mixed with BCA reagent (from Interchim) and incubated at 37° C. for 45 min.
  • the protein content was determined at 562 nm against a calibration curve (BSA) in the concentration range of the assay used.
  • TLC Film Silica Gel 60 (Merck). Authentic substances were applied as reference. One end of the TLC film was dipped in solvent until the top of the mobile phase was reached. The TLC film was dried and was developed with phosphomolybdic acid.
  • 7 ⁇ -HSDH coding sequences were PCR-amplified.
  • the PCR products were obtained using the genomic DNA of Collinsella aerofaciens ATCC 25986 (DSM 3979) as template and the primers 5′-gggaattc CATATG AACCTGAGGGAGAAGTA-3′ (SEQ ID NO:3) and 5′-ccc AAGCTT CTAGTCGCGGTAGAACGA-3′ (SEQ ID NO:4).
  • the NdeI and HindIII cleavage sites in the primer sequences are underlined.
  • the PCR product was purified using the PCR-Purification-Kit (Qiagen) and then cut with the enzymes NdeI and HindIII.
  • the corresponding vector was also cut with NdeI and HindIII.
  • the products were applied to agarose gel, separated, cut out and purified.
  • the cut PCR product and the cut vector were ligated by means of T4-ligase.
  • the ligant was transformed into E. coli DH5 ⁇ .
  • the resultant vector (contains the gene of 7 ⁇ -HSDH) was confirmed by sequencing and transformed into E. coli BL21(DE3) and induced with IPTG and expressed.
  • Expression was carried out in 50 ml LB medium.
  • a colony on LB-agar plate in 5 ml LB medium (contains corresponding antibiotics) was picked and incubated overnight at 37° C. and 160 rpm.
  • the 50 ml LB medium (contains corresponding antibiotics) was inoculated with 500 ⁇ l of preculture.
  • the culture was incubated at 37° C. and 160 rpm. Up to OD600 approx. 0.8, expression was induced by adding 0.5 mM IPTG. After 6 h or overnight, the cells were centrifuged off. The pellets were resuspended in 6 ml potassium phosphate buffer (50 mM, pH 8, contains 0.1 mM PMSF) and disrupted with ultrasound. The cell debris was removed by centrifugation.
  • 6 ml potassium phosphate buffer 50 mM, pH 8, contains 0.1 mM PMSF
  • 7 ⁇ -HSDH showed activity of 60 U/ml against UDCA, activity of 35 U/ml against 7-keto-LCA and activity of 119 U/ml against DHCA in the presence of NADP + or NADPH.
  • the activity against CA is not detectable.
  • the gene that codes for 7 ⁇ -HSDH was subcloned into pET28a+ with His-Tag, to permit rapid purification.
  • This 70-HSDH with His-Tag was actively expressed in E. coli BL21(DE3) as described above. Purification was carried out with a Talon column. The column was first equilibrated with potassium phosphate buffer (50 mM, pH 8, with 300 mM NaCl). After loading of the cell lysate, the column was washed with potassium phosphate buffer (50 mM, pH 8, with 300 mM NaCl). The 7 ⁇ -HSDH was eluted with potassium phosphate buffer (50 mM, pH 8, with 300 mM NaCl and 200 mM imidazole). The imidazole in the eluate was removed by dialysis. The yield in purification was 76% with purity of approx. 90%.
  • the gene coding for 7 ⁇ -HSDH was once again amplified from the genomic DNA by PCR and using primers, as described above for production example 1:
  • the PCR product was once again purified as described above and digested with the restriction endonucleases NdeI and HindIII.
  • the digested PCR product was purified again and cloned into the pET-28a(+) vector using the T4-ligase, to produce an expression vector.
  • the resultant expression construct was then transformed into E. coli DH5 ⁇ cells.
  • the protein to be expected should have 20 amino acid residues comprising a signal peptide and an N-terminal 6 ⁇ His-Tag and a thrombin cleavage site. The sequence of the inserted DNA was verified by sequencing.
  • E. coli BL21(DE3) was transformed with the expression construct.
  • the E. coli BL21(DE3) strain containing the expression construct was multiplied in LB medium (2 ⁇ 400 ml in 2-liter shaking bottles) containing 30 ⁇ g/ml kanamycin.
  • the cells were harvested by centrifugation (10.000 ⁇ g, 15 min, 4° C.).
  • the pellet was resuspended in 20 ml phosphate buffer (50 mM, pH 8, containing 0.1 mM PMSF).
  • the cells were disrupted by ultrasound treatment for 1 minute with constant cooling (40 W power, 40% working interval and 1 min pause) using Sonifier 250 ultrasonic equipment (Branson, Germany). Lysis was repeated three times.
  • the cell extract was centrifuged (22.000 ⁇ g, 20 min, 4° C.). The supernatant was loaded on a Talon column (Clontech, USA), equilibrated with loading buffer (50 mM potassium phosphate, 300 mM NaCl, pH 8). The procedure was carried out at 24° C. Unbound material was washed away by washing the column with loading buffer (3 column volumes). Weakly binding protein was removed by washing with washing buffer (20 mM imidazole in the loading buffer; 3 column volumes). The His-Tag-7 ⁇ -HSDH protein was eluted with elution buffer (200 mM imidazole in the loading buffer).
  • the eluate was dialyzed overnight in a dialysis tube with a molecular exclusion limit of 5 kDa (Sigma, USA) in 2 liters of potassium phosphate buffer (50 mM, pH 8) at 4° C. Finally the sample was transferred to a new tube and stored at ⁇ 20° C. for further analysis.
  • the protein concentration was determined using a BCA Test Kit (Thermo, USA) according to the manufacturer's instructions.
  • the sample was analyzed by 12.5% SDS-PAGE and staining with Coomassie Brilliant Blue. The purity of the protein was determined densitometrically using Scion Image Beta 4.0.2 (Scion, USA).
  • the molecular weight of 7 ⁇ -HSDH was determined by comparing its elution volume with that of protein standards (serum albumin (66 kDa), ⁇ -amylase from Aspergillus oryzae (52 kDa), pig pancreas trypsin (24 kDa) and hen's egg lysozyme (14.4 kDa)).
  • protein standards serum albumin (66 kDa), ⁇ -amylase from Aspergillus oryzae (52 kDa), pig pancreas trypsin (24 kDa) and hen's egg lysozyme (14.4 kDa)).
  • the reaction mixture for the enzyme assay contained, in a total volume of 1 ml, 50 ⁇ mol potassium phosphate (pH 8), 0.1 ⁇ mol NAD(P)H or NAD(P) + , substrates and protein.
  • the reaction mixture was contained in cuvettes with a light path length of 1 cm.
  • the 7 ⁇ -HSDH activity was determined by recording the variation in NAD(P)H concentration against the extinction at 340 nm using a spectrophotometer (Ultraspec 3000, Pharmacia Biotech, Great Britain).
  • the enzyme activities were determined as enzyme units (U, i.e. ⁇ mol/min) using the molar extinction coefficient of 6.22 mM ⁇ 1 ⁇ cm ⁇ 1 at 25° C.
  • Several different measurements were performed with the variables substrate, coenzyme, concentration, pH, buffer and incubation temperature. The kinetic constants were determined using standard methods.
  • a fusion protein provided with a His-Tag on the N-terminus was obtained with a 7 ⁇ -HSDH yield of 332.5 mg (5828 U) per liter of culture.
  • the 7 ⁇ -HSDH provided with the His-Tag was purified in one step by immobilized metal ion affinity chromatography (purity >90%, yield 76%, cf. FIG. 2 .).
  • the main bands of lanes 1 and 2 represent the expected expression product at 30 kDa, which corresponds to the predicted molecular weight derived from the amino acid sequence of the gene. However, a molecular weight of 56.1 kDa is found for 7 ⁇ -HSDH by gel filtration. This proves the dimeric nature of the 7 ⁇ -HSDH from Collinsella aerofaciens DSM 3979.
  • the amino acid sequence of the 7 ⁇ -HSDH used according to the invention was compared with known HSDH sequences (alignment not shown).
  • the observed sequence similarity indicates that the enzyme according to the invention belongs to the family of short-chain dehydrogenases (SDR).
  • SDRs short-chain dehydrogenases
  • SDRs have very low homology and sequence identity (Jornvali, H., B. Persson, M. Krook, S. Atrian, R. Gonzalez-Duarte, J. Jeffery, and D. Ghosh. 1995. Short-chain dehydrogenases/reductases (SDR). Biochemistry 34: 6003-13 and Persson, B., M. Krook, and H. Jornvall. 1991. Characteristics of short-chain alcohol dehydrogenases and related enzymes.
  • 7 ⁇ -HSDHs from Clostridium sordellii, Brucella melitensis and Escherichia coli belong to the same subgroup.
  • the two 3 ⁇ -HSDHs show a more pronounced similarity than other HSDHs.
  • the prokaryotic 7 ⁇ -HSDH is related to the animal 113-HSDH subgroup, comprising Cavia porcellus, Homo sapiens and Mus musculus.
  • the 7 ⁇ -HSDH activity was determined with purified enzyme for various substrates as a function of the pH.
  • an optimal activity was observed in the range from pH 9 to 10 with gradual decline on the acidic side.
  • Different buffers only have a slight influence on the activity of 7 ⁇ -HSDH at identical pH.
  • the NADP-dependent 7 ⁇ -HSDH used according to the invention shows the following stability behavior: after 400 min the activity at 30° C. was about 30% lower than at 23° C. At 30° C., the enzyme was completely inactivated after 1500 min, whereas at 23° C. and 1500 min the residual activity was 20%. No significant activity loss was observed during storage at ⁇ 20° C. in potassium phosphate buffer (50 mM, pH 8) over a period of some months after repeated freezing and thawing.
  • Position 39 of the amino acid sequence (comprising start methionine) (cf. SEQ ID NO:2) was mutated.
  • the mutagenesis primers stated below were used for the site-directed mutagenesis of 7 ⁇ -HSDH.
  • the primers were selected based on the 7 ⁇ -HSDH gene sequence, so that they bring about the desired amino acid exchange. It was borne in mind that the base to be mutated is localized centrally in the primer, and that the melting points of the primer pairs are in the same region.
  • the primer pair 7beta_mut_G39A_fwd and 7beta_mut_G39A rev was used for preparing the G39A mutant.
  • the primer pair 7beta_mut_G39S_fwd and 7beta_mut_G39S_rev was used for preparing the G39S mutant.
  • N6-adenine-methylated, double-stranded plasmid DNA which bears the gene to be mutated, serves as template.
  • N6-adenine-methylated plasmid DNA is isolated from dam + E. coli strain such as for example E. coli DH5.
  • the polymerase chain reaction is carried out as described above.
  • the primers are lengthened complementarily to the template, so that plasmids with the desired mutation are formed, which have a strand break.
  • the increase in DNA yield is only linear, as newly formed DNA molecules cannot serve as template for the PCR reaction.
  • the parental, N6-adenine-methylated DNA is digested by the restriction enzyme DpnI.
  • DpnI restriction enzyme
  • This enzyme has the particular feature that it restricts nonspecifically N6-adenine-methylated DNA, but not the newly formed, nonmethylated DNA. Restriction was carried out by adding 1 ⁇ L DpnI to the PCR reaction mixture and incubating for 1 h at 37° C.
  • a cofactor concentration of 100 ⁇ M NADPH relative to the reaction volume was used, while the substrate concentration was varied over a range from 7 ⁇ M to 10 mM dehydrocholic acid with 31 different concentrations (in each case relative to the reaction mixture).
  • the reaction rate was determined by linear regression over the first 4 measurements (0 s-18 s).
  • V max , K m and K i were determined by nonlinear regression.
  • v max are roughly 1.5 times the value of the wild-type protein (21.0 ⁇ 1.1 U/mg for the G39A mutant and 20.1 ⁇ 1.0 U/mg for the G38S mutant versus 14.6 ⁇ 1.0 U/mg for the wild-type protein).
  • the v max values are not to be regarded as maximum attainable specific enzyme activities, since with increasing substrate concentration the specific enzyme activities do not approach v max asymptotically, but decrease again.
  • the specific enzyme activities at the maxima of the kinetic curves are ⁇ 13 U/mg for the wild-type protein and ⁇ 19 U/mg (G39A) and ⁇ 20 U/mg(G39S) for the mutant proteins.
  • the enzyme activities that can be reached at the substrate concentrations used for whole-cell biotransformation are more interesting than the maximum attainable enzyme activities. As an example, these are to be compared for the substrate concentration of 10 mM dehydrocholic acid, and are ⁇ 6.7 U/mg for the wild-type protein, ⁇ 13 U/mg for the G39A mutant and ⁇ 18 U/mg for the G39S mutant.
  • the G39A mutant At 10 mM substrate concentration, the G39A mutant would accordingly be twice as active as the wild-type protein, and the G39S mutant would even have about 3 times its activity. These differences should be even more pronounced at higher substrate concentrations, as the wild-type protein displays stronger substrate inhibition than the G39A mutant, and the G39A mutant is in its turn more strongly substrate-inhibited than the G39S mutant.
  • the template used for the amplification is genomic DNA of Mycobacterium vaccae , which was obtained from the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, DMSZ), Brunswick.
  • the primers for the amplification were fdh_for (5′-CGAT CATATG GCAAAGGTCCTGTGCGTTC-3′) (SEQ ID NO:23) and fdh_rev (5′-GCTA GAATTC TCAGCCGCCTTCTTGAACT-3′) (SEQ ID NO:24), obtained from Eurofins MWG GmbH, Ebersberg.
  • the recognition sites for the restriction enzymes are underlined.
  • the rev-primer contains the EcoRI cleavage site and the for-primer contains the NdeI cleavage site.
  • the bands of the correct size (1.2 kb for the FDH-gene, 5.4 kb for the pET21a(+)-plasmid) were cut out of the agarose gel with a scalpel and were isolated using the QIAquick Gel Extraction Kit (QIAGEN, Hilden) according to the manufacturer's protocol.
  • the 10 ⁇ L ligation preparation is added to 200 ⁇ l of chemically competent E. coli DH5 ⁇ prepared according to the standard protocol. Next there is a 30-min incubation step on ice, followed by heat shock at 42° C. (90 seconds). Then 600 ⁇ l of sterile LB medium is added to the transformation preparation and the cells are incubated at 200 rpm and 37° C. in a shaking incubator for 45 minutes.
  • the preparation is centrifuged at 3000 rpm for 60 seconds in a benchtop centrifuge, 700 ⁇ l of the supernatant is discarded, the cells are resuspended in the remaining supernatant and plated out on an LB-agar plate with 100 mg/l ampicillin. The agar plate is then incubated overnight at 37° C.
  • Aspartate (D) 221 is an amino acid with a negatively-charged large side chain, which is located directly next to the arginine (R) residue, by which NADP + is to be bound. This can lead to repulsion of the phosphate group in the NADP 4 , which is also negatively charged. The aspartate is therefore replaced with the small uncharged amino acid residue glycine (G).
  • Plasmid pET21a(+)FDH was used as template.
  • the PCR program used is shown in the following table:
  • the length of the megaprimer becomes 650 bp.
  • gel electrophoresis and isolation of the desired band from the gel are carried out.
  • a second PCR is carried out as whole plasmid PCR with the megaprimers as primers and the plasmid DNA (pETfdh) as template.
  • the reaction mixture and the temperature scheme for the whole plasmid PCR are shown in the following tables.
  • the 2 ⁇ EZClone enzyme mix, the EZClone solution 1, the 1.1 kb marker and the DpnI were obtained from the GeneMorph II EZClone Domain Mutagenesis Kit (Stratagene).
  • the first step in the PCR program (68° C., 5 min) is for removing the bases appended nonspecifically by the Taq-polymerase by the 3′->5′ exonuclease activity of the polymerase used in the MEGA WHOP PCR.
  • the PCR product is a double-stranded plasmid with single-strand breaks, which are only closed in E. coli.
  • 10 U DpnI were added to the 50 ⁇ L PCR product and the preparation was incubated at 37° C. for two hours. DpnI only degrades methylated DNA, i.e. the template DNA used, but not the megaprimer or the synthesized plasmid.
  • the template plasmid must be produced with a dam + strain (such as DH10B or JM109), to obtain methylated starting DNA.
  • LB preculture was inoculated from frozen stored material or with a colony from an agar plate and the preculture was incubated overnight. Incubation was carried out at 37° C. and 250 rpm. The OD of the preculture was determined and the culture was inoculated to an OD of 0.1. On reaching an OD of 0.5-1 (after about 2.5 h), it was induced with IPTG (final concentration 1 mM). The cells were harvested three hours after induction.
  • the cells were disrupted mechanically with glass beads. For this, 0.5 mL of glass beads were added to 0.5 mL of cell sample in a 1.5-mL reaction vessel and the reaction vessel was shaken for 3 min at maximum frequency (30 s ⁇ 1 ) in a Retsch vibratory mill. After cell maceration, the glass beads were centrifuged off (2 min, 13000 rpm). Before and after maceration, the culture was put on ice, to minimize protein denaturation through heating of the sample in the vibratory mill. This maceration protocol is optimized for the use of samples frozen at ⁇ 20° C.
  • K M (mM) K M (mM) Enzyme pH Substrate Cofactor (U ⁇ mg ⁇ 1) substrate cofactor FDH 6 Sodium formate NAD + 4.6 46.86 0.83 6 Sodium formate NADP + 1 0.3 7 Sodium formate NAD + 5.1 45.56 0.61 7 Sodium formate NADP + 1.2 0.51 8 Sodium formate NAD + 4.7 59.26 0.52 8 Sodium formate NADP + 1 1.01 The data prove that the mutant produced can utilize both NAD + and NADP + as cofactor.
  • the target is deletion of the disturbing 7 ⁇ -HSDH activity in the expression strain E. coli BL21 (DE3).
  • an antibiotic resistance gene is inserted in the target gene of 7 ⁇ -HSDH, so that the target gene is switched off.
  • the knockout mutant was prepared using the kit TargeTronTM Gene Knockout System from Sigma Aldrich according to the manufacturer's instructions.
  • the QIAquick PCR Purification Kit from Qiagen was used for purifying the PCR product according to step B.6. of the TargeTronTM Gene Knockout System.
  • Ligation of the HindIII/BsrGI-digested intron PCR product into the linearized pACD4K-C vector was carried out as follows: the reaction was carried out overnight at 16° C.
  • the transformation preparations were plated out on LB-agar plates, containing 33 ⁇ g/mL kanamycin. Kanamycin-resistant cells were picked and these were in each case inoculated over several nights in 5 ml LB overnight cultures (in each case with 5 ⁇ l of a kanamycin solution (33 mg/ml)). Finally a 200 ml LB culture (with 200 ⁇ l kanamycin solution (33 mg/mL)) was inoculated with an overnight culture and was incubated for 5 h at 37° C. and 180 rpm in a shaking incubator. Then the temperature was raised to 42° C. for 1 hour.
  • This culture was used for inoculating a 5 mL LB overnight culture (in each case with 5 ⁇ l of a kanamycin solution (33 mg/mL)). After incubation overnight at 37° C. and 180 rpm, the culture was streaked on an LB-agar plate with 33 ⁇ g/mL kanamycin. After overnight incubation at 37° C., colonies were picked and streaked on LB-agar plate with 33 ⁇ g/mL kanamycin and 34 ⁇ g/mL chloramphenicol.
  • chloramphenicol-sensitive mutants were found. This is necessary in order to confirm the loss of the plasmid that is carried by the inducible knockout system and is no longer required after successful knockout.
  • the 7 ⁇ -HSDH gene was amplified by colony PCR with the primers 7alpha-ko-check_fwd (5′-TTAATTGAGCTCCTGTACCCCACCACCACC-3′) SEQ ID NO:32 and 7alpha-ko-check_rev (5′-GTGTTTAATTCTGACAACCTGAGACTCGAC-3′) SEQ ID NO:33.
  • the resultant fragment had a length of approx. 2.5-3 kb and was sequenced with the primer 7alpha-ko-check_fwd.
  • the sequencing showed that the DNA sequence of 7 ⁇ -HSDH is interrupted by an insert from the pACD4K vector, resulting in knockout of 7 ⁇ -HSDH (sequencing data not shown).
  • the 20 ml reaction mixture contains 50 mM 7-keto-LCA (approx. 0.4 g), 5 U/ml 7 ⁇ -HSDH and 0.05 mM NADP + . 4 U/ml ADH and 1% isopropanol were used for regeneration of NADPH (see Scheme 1).
  • the reaction was carried out in a fume cupboard at pH8 and 24° C. with stirring. As acetone evaporates more quickly than isopropanol, the reaction is shifted toward formation of UDCA. Further 1% isopropanol was added after 24 h, 48 h and 72 h.
  • the product was analyzed by TLC (silica gel 60, Merck, solvent petroleum ether and ethyl acetate 1:10, vol:vol).
  • TLC sica gel 60, Merck, solvent petroleum ether and ethyl acetate 1:10, vol:vol.
  • the product was compared with authentic references 7-keto-LCA, UDCA and CDCA.
  • the TLC analysis shows that UDCA was formed from 7-keto-LCA by the 7 ⁇ -HSDH.
  • the enantiomer CDCA is not detectable in TLC.
  • the intermediate 3,12-diketo-7 ⁇ -CA (produced according to reaction example 2) was transformed further by 3 ⁇ -HSDH (SEQ ID NO:5 and 6) from Comamonas testosteroni (Mobus, E. and E. Maser, Molecular Cloning, overexpression, and characterization of steroid - inducible 3 alpha - hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni.
  • Comamonas testosteroni Manton, E. and E. Maser, Molecular Cloning, overexpression, and characterization of steroid - inducible 3 alpha - hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni.
  • a novel member of the short - chain dehydrogenase/reductase superfamily J Biol Chem, 1998. 273(47): p. 30888-96
  • This 3 ⁇ -HSDH requires cofactor NADH, which was regenerated by the FDH (see FIG. 3 ).
  • 4 U/ml 3 ⁇ -HSDH, 1 U/ml FDH (NADH-dependent, Codexis), 200 mM sodium formate and 0.05 mM NAD + were added to the reaction mixture.
  • the product was acidified with 2 M HCl to pH2 and extracted with 6 ⁇ 10 ml ethyl acetate. After evaporation, 1.07 g of product was obtained.
  • the product 12-keto-UDCA was analyzed and confirmed by TLC and NMR.
  • 3alpha-HSDH was prepared as for the preparation of 7 ⁇ -HSDH, but with the plasmid pET22b+, and was used without further purification.
  • the purpose of this example is to investigate whether a two-step enzymatic conversion of DHCA to 12-keto-UDCA with simultaneous cofactor regeneration with an FDH mutant used according to the invention is possible.
  • the FDH mutant D221G used accepts both NADP + and NAD + as cofactor, for the NADH-dependent 3 ⁇ -HSDH it is not necessary for the reaction mixture to contain any additional cofactor regeneration system.
  • FDH* designates the mutant FDH D221G.
  • the enzymes 7 ⁇ -HSDH from Collinsella aerofaciens, 3 ⁇ -HSDH from Comamonas testosteroni and the FDH mutant D221G derived from FDH from Mycobacterium vaccae were expressed separately from one another in a modified E. coli expression strain and were used for the reaction.
  • the E. coli strain used for expression was modified so that it does not express any 7 ⁇ -HSDH enzyme activity.
  • This side activity, widely occurring in E. coli can lead, in reactions of the present type (stereospecific conversion of the 7-keto group), to undesirable side reactions and therefore to contamination of the reaction product to be produced.
  • Plasmids for the expression of 7 ⁇ -HSDH, 3 ⁇ -HSDH and FHD D221G Plasmids for the expression of 7 ⁇ -HSDH, 3 ⁇ -HSDH and FHD D221G:
  • FDH mutant 47 U (for sodium formate and NAD + ). Content and purity of the proteins were determined by SDS-PAGE and densitometer scanning using Scion Image Beta 4.0.2 (Scion, USA). 6.4 Preparative-Scale Enzymatic Synthesis of 12-keto-UDCA
  • the product was analyzed by HPLC and NMR.
  • the aim was to investigate whether a two-step whole-cell reduction of dehydrocholic acid (DHCA) to 12-keto-ursodeoxycholic acid (12-keto-UDCA) (cf. scheme according to reaction example 6, above) is possible.
  • DHCA dehydrocholic acid
  • 12-keto-ursodeoxycholic acid (12-keto-UDCA)
  • the knockout strain E. coli BL21 (DE3) hdhA ⁇ KanR + pET21a(+) FDH 7 ⁇ -HSDH pCOLA(mod) 3 ⁇ -HSDH prepared above was used, in which, in addition to the 7 ⁇ -HSDH and the mutant FDH D221G, a 3 ⁇ -HSDH from Comamonas testosteroni is expressed recombinantly.
  • the FDH mutant used accepts both NADP + and NAD + as cofactor, for the NADH-dependent 3 ⁇ -HSDH it was not necessary to insert any additional cofactor regeneration system into the biotransformation strain.
  • the polymerase chain reaction was used for cloning the 7 ⁇ -HSDH.
  • the plasmid pET22b(+) 7 ⁇ -HSDH served as template for amplification of the 70-HSDH.
  • PCR reactions were carried out in 500 ⁇ L PCR tubes with 20 ⁇ L reaction volume. The reactions were carried out in a Thermocycler from the company Eppendorf. For amplification of 7 ⁇ -HSDH, in each case 4 ⁇ L HF-buffer, 1 ⁇ L template DNA, 1 ⁇ L each of forward and reverse primer (10 ⁇ M), 0.4 ⁇ L of deoxynucleotide triphosphate solution (10 mM) and 0.2 ⁇ L of Phusion DNA polymerase (2 U/ ⁇ L) were added. The volume of the preparation was adjusted to 20 ⁇ L with RNase-free water.
  • DNA fragments For purification of DNA fragments, first they were separated by agarose gel electrophoresis. The corresponding bands were made visible with UV light, identified based on their size and cut out of the gel with a scalpel. Extraction was carried out with the QIAquick Gel Extraction Kit according to the manufacturer's protocol. 30-50 ⁇ L H 2 O was used for elution of the purified DNA.
  • the restriction reactions were carried out in a total volume of 20-50 ⁇ L. For this, 10-20 U of the respective restriction enzymes was added to the DNA to be cut. In addition, the reaction buffer recommended by the manufacturer was used and optionally 0.5 ⁇ L of bovine serum albumin (BSA, 10 mg/mL) was added, based on the manufacturer's recommendation. Restriction digestion was carried out for 2 h at 37° C. Then the restricted fragments were purified either by gel extraction (vector digestion products) or using the QIAquick PCR Purification Kit (digested PCR products).
  • BSA bovine serum albumin
  • DNA was purified using the QIAquick PCR Purification Kit. Purification was carried out according to the manufacturer's instructions, using 30-50 ⁇ L H 2 O for elution of the purified DNA.
  • both DNA molecules were cut with the same restriction enzymes.
  • the enzyme used was T4-DNA-ligase, which catalyzes the formation of a phosphodiester bond between a free 5′-phosphate group and a free 3′-OH end of a deoxyribonucleic acid.
  • 7 ⁇ -HSDH was cloned into pET21a(+) FDH, so that the 7beta-HSDH coding gene is located downstream of the FDH.
  • a stop codon had to be inserted in the FDH at the 5′-end.
  • the C-terminal valine residue was replaced with an alanine residue and a further three amino acids were appended, resulting in the following C-terminal sequence: Lys-Lys-Ala-Ala-Gly-Asn-Ser-stop.
  • an additional ribosomal binding site was inserted in 7 ⁇ -HSDH between FDH and 7beta-HSDH.
  • the primers 7beta_fwd_EcoRI and S — 7beta_rev_HindIII were used for PCR amplification of 7 ⁇ -HSDH.
  • the enzymes 7 ⁇ -HSDH and 3 ⁇ -HSDH must also be present in a cell.
  • the vector construct pCOLA(mod) 3 ⁇ -HSDH a modified derivative of the pCOLA-duet vector, was prepared, which is cotransformable with pET-vectors.
  • the 3 ⁇ -HSDH coding gene was cut out via the NdeI and BlpI cleavage sites from the vector pET22b(+) 3 ⁇ -HSDH and, via the same cleavage sites, cloned into MCS2 of the pCOLA(mod) vector.
  • Both vectors were cotransformed into the aforementioned strain, as described below.
  • the genes are IPTG-inducible.
  • E. coli BL21 (DE3) hdhA ⁇ KanR + or DH5 ⁇ were thawed and 1 ⁇ L DNA was added. These were first incubated on ice for 45 min. Then the cells were submitted to heat shock for 45 s at 42° C. Then 600 ⁇ L of LB medium was added to the cells and they were shaken at 37° C. and 200 rpm, so that they could develop the desired antibiotic resistance. Next the cells were centrifuged in a benchtop centrifuge at 3000 rpm for 2 min, after which 150 ⁇ L of the supernatant was discarded. The cells were resuspended in the remaining supernatant and plated out on LB-agar plates with the corresponding antibiotic. Then the plates were incubated overnight at 37° C.
  • the corresponding E. coli BL21 (DE3) hdhA ⁇ KanR + comprising the two expression plasmids was incubated overnight at 37° C. and 200 rpm in 5 mL of LB medium with addition of the corresponding antibiotic. Then 200 mL of TB medium with addition of the corresponding antibiotic was inoculated with this preculture and incubated at 37° C., 250 rpm. On reaching OD600 of 0.6-0.8, expression of the recombinant protein was induced with 1 mM isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) and the culture was incubated at 25° C., 160 rpm for a further 21 h.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the chromatograms of the HPLC measurement are shown in FIG. 7 .
  • the HPLC analysis was carried out as follows: The chromatography column used was the reverse-phase chromatography column Hi-bar Purospher 125-4 RP-18e (5 ⁇ m) from the company Merck, Darmstadt. In this case a nonpolar phase serves as stationary phase, whereas a polar phase forms the mobile phase.
  • the method of gradient elution was used for the HPLC analysis. An increasing proportion of acetonitrile is added to the eluent, phosphoric acid water, (pH 2.6), and the acidification of the solvent causes a uniform degree of protonation of the analytes to be investigated. After elution of all components, the chromatography column is equilibrated with the starting ratio of the two solvents.
  • Gradient elution was carried out by first pumping a solvent mixture with a constant composition of 65% (v/v) of phosphoric acid water and 35% (v/v) of acetonitrile through the HPLC column for 3 min. From minute 3 to 7.5, the proportion of acetonitrile is increased linearly to 39% (v/v). Between minute 7.5 and 10 there is another linear increase of the proportion of acetonitrile to 40% (v/v). For elution of all sample components, the proportion of acetonitrile is increased between minute 10 and 11 also linearly to 70% (v/v) and held constantly at this value for a further two minutes.
  • the aim was to investigate whether a two-step whole-cell reduction of dehydrocholic acid to 12-keto-ursodeoxycholic acid in a cellular single-plasmid system is possible.
  • the plasmid construct pET21a(+) FDH 7 ⁇ -HSDH(G39A) 3 ⁇ -HSDH ( FIG. 8 ) is prepared as follows: Starting from the plasmid pET21a(+) FDH 7 ⁇ -HSDH the wild-type 3 ⁇ -HSDH ( C. testosteroni ; SEQ ID NO: 5,6) was cloned in after 7 ⁇ -HSDH via the HindIII and NotI cleavage sites. For amplification of 3 ⁇ -HSDH, the primers 3alpha_fwd_HindIII and 3alpha_rev_NotI were used, with the plasmid pET22b(+) 3 ⁇ -HSDH serving as template for this.
  • the G39A mutation was inserted into 7 ⁇ -HSDH by site-directed mutagenesis according to the QuikChange protocol.
  • the mutagenesis primers 7beta_mut_G39A_fwd and 7beta_mut_G39A_rev were used.
  • the plasmid prepared was transformed into E. coli BL21 (DE3) hdhA ⁇ KanR + .
  • E. coli BL21 (DE3) hdhA ⁇ KanR + pET21a(+) FDH 7 ⁇ -HSDH(G39A) 3 ⁇ -HSDH whole-cell reactions were carried out under the following conditions at the 150-mL scale: cell density OD 30, 50 mM DHCA, 750 mM sodium formate, suspended in 50 mM potassium phosphate buffer (pH 6.5) as cell and substrate suspension.
  • the reactions were carried out for 3.5 h at 25° C.
  • the results of the HPLC analysis (for procedure see reaction example 7, above) are shown in FIG. 9 .
  • One possibility for generating an NADH-specific 7 ⁇ -HSDH consists of modifying the available enzyme by various techniques of mutagenesis. Therefore, using site-directed mutagenesis, individual amino acids of 7 ⁇ -HSDH were to be substituted with others, which cause change of the cofactor specificity of 7 ⁇ -HSDH from NADPH to NADH, so that advantageously NADP can be replaced with the less expensive NAD.
  • the 7 ⁇ -HSDH mutants G39D, G39D/R40L, G39D/R40I and G39D/R40V were produced.
  • the G39D mutant is produced by Quickchange mutagenesis, and the other mutants are produced by the PCR method of Sanchis et al. (Sanchis J, Fernández L, Carballeira J D, Drone J, Gumulya Y, Höbenheim H, Kahakeaw D, Kille S, Lohmer R, Peyralans J J, Podtetenieff J, Prasad S, Soni P, Taglinger A, Wu S, Zilly F E, Reetz M T.
  • G39D 7beta mut G39D fwd (SEQ ID NO: 41): CGTCGTCATGGTC GACC GTCGCGAGG 7beta mut G39D rev (SEQ ID NO: 42): CCTCGCGACG GTC GACCATGACGACG G39D R40L 7beta mut G39D R40L fwd (SEQ ID NO: 43): CGTCGTCATGGTC GACCTG CGCGAGG 3alphamut_AntiMid_rev (SEQ ID NO: 44): CCGCCGCATCCATACCGCCAGTTGTTTACCC G39D R40I 7beta mut G39D R40I fwd (SEQ ID NO: 45): CGTCGTCATGGTC GACATT CGCGAGG 3alphamut_AntiMid_rev (SEQ ID NO: 44): CCGCCGCATCCATACCGCCAGTTGTTTACCC G39D R40V 7beta mut G39D R40
  • the mutants prepared are evaluated by means of enzyme-kinetic investigations, the results of which are shown as graphs in FIG. 11 . 0 . 1 mM NADPH is used as cofactor for investigating the unmodified 7 ⁇ -HSDH, but 0.5 mM NADH is used for investigating the 7 ⁇ -HSDH mutants.
  • the need to increase the cofactor concentration in the enzyme-kinetic investigation of the 7 ⁇ -HSDH mutants is due to the increased semisaturation concentrations for the cofactor NADH in the mutants.
  • Equation 1 (Michaelis-Menten Equation)
  • EA x specific enzyme activity
  • mol L ⁇ 1 K m semisaturation concentration
  • mol Equation 2 Mathaelis-Menten Equation with Substrate Inhibition
  • EA x v max ⁇ c s K m + ( 1 + c s K i ) ⁇ c s
  • EA x specific enzyme activity
  • U ⁇ mol min ⁇ 1 mg ⁇ 1 c
  • mol L ⁇ 1 K m semisaturation concentration
  • mol L ⁇ 1 K i inhibition constant
  • mol L ⁇ 1 The following table gives enzyme-kinetic parameters of the unmodified wild-type 7 ⁇ -HSDH and the mutants thereof with altered cofactor specificity. NADPH is used as cofactor for the wild type, whereas NADH is used as cofactor for the mutants.
  • the mutagenesis primers shown below were used for the site-directed mutagenesis of 7 ⁇ -HSDH.
  • the primers were selected on the basis of the 7 ⁇ -HSDH gene sequence, so that they bring about the desired amino acid exchange. It was noted that the base to be mutated is localized centrally in the primer, and that the melting points of the primer pairs are located in the same region.
  • N6-adenine-methylated, double-stranded plasmid DNA which carries the gene to be mutated, serves as template.
  • N6-adenine-methylated plasmid DNA is isolated from dam + E. coli strain, for example E. coli DH5.
  • the polymerase chain reaction is carried out as described above.
  • the primers are lengthened complementarily to the template, so that plasmids with the desired mutation are formed, which have a strand break.
  • the increase in DNA yield is in this case only linear, as newly formed DNA molecules cannot serve as template for the PCR reaction.
  • the PCR product was purified using a PCR-Purification-Kit (Analytik Jena) and the parental, N6-adenine-methylated was digested with the restriction enzyme DpnI.
  • This enzyme has the particular characteristic that it restricts N6-adenine-methylated DNA nonspecifically, but not the newly formed, nonmethylated DNA. Restriction was carried out by adding 1 ⁇ L. DpnI to the PCR reaction mixture and incubating for 2 h or overnight at 37° C.
  • the mutation was introduced into 7 ⁇ -HSDH by site-directed mutagenesis according to the QuikChange protocol.
  • the mutagenesis primers from the table shown in Section 7.1 were used.
  • the 7 ⁇ -HSDH mutants together with an NADH-dependent formate dehydrogenase from Mycobacterium vaccae were inserted in an expression vector.
  • the vector into which 7 ⁇ -HSDH (G39D) is inserted bears the designation pFr7(D), corresponding to SEQ ID NO:49; the vector into which 7 ⁇ -HSDH (G39D R40L) is inserted bears the designation pFr7(DL), corresponding to SEQ ID NO:50; the vector into which 76-HSDH (G39D R40I) is inserted bears the designation pFr7(DI), corresponding to SEQ ID NO:51, and the vector into which 7 ⁇ -HSDH (G39D R40V) is inserted bears the designation pFr7(DV), corresponding to SEQ ID NO:52.
  • a general plasmid map of these vectors is shown in FIG. 13 .
  • the reaction mixtures for the biotransformations contained 17.7 g L ⁇ 1 BTM whole-cell biocatalysts, 100 mM substrate (DHCA), 500 mM sodium formate and are suspended in 50 mM potassium formate buffer (pH 6.5). The process took place at the 20 mL scale as a batch process without pH monitoring. The concentrations of product and substrate after 24 h are shown in FIG. 14 . It can be seen that all mutants are suitable for the whole-cell biocatalytic conversion of DHCA to 3,12-diketo-UDCA.
  • NADH-dependent 7 ⁇ -HSDH in the two-step whole-cell biocatalytic conversion of DHCA to 12-diketo-UDCA is to be demonstrated below.
  • the mutants 7 ⁇ -HSDH (G39D) together with an NADH-dependent formate dehydrogenase from Mycobacterium vaccae and an NADH-dependent 3 ⁇ -HSDH from Comamonas testosteroni were inserted into an expression vector.
  • the use of 7 ⁇ -HSDH (G39D) is shown as an example for all NADH-dependent 7 ⁇ -HSDHs.
  • the vector bears the designation pFr3T7(D) and is shown schematically in FIG. 15 .
  • E. coli BL49 (identical to E. coli BL21(DE3) hdhA ⁇ KanR + ); the resultant strain bears the designation E. coli BL49 pFr3T7(D).
  • This strain was cultured in the 7 L bioreactor at a working volume of 4 L in the defined minimal medium. A brief initial phase at 37° C. was followed by a substrate-limited exponential growth phase at 30° C. On reaching an optical density of 25-30, protein expression of the cells was induced by adding 0.5 mM IPTG. Expression was then carried out for 24 h at 20° C. The cells (32.0 g L ⁇ 1 BTM ) were harvested by centrifugation, resuspended in potassium phosphate buffer (pH 6.5) and stored according to the standard protocol at ⁇ 20° C.
  • Biotransformation was set up at the 20-mL scale in the following reaction conditions: 100 mM DHCA, 17.7 g L ⁇ 1 BTM of the stored biocatalyst, 500 mM ammonium formate, 26% glycerol, 50 mM MgCl 2 , suspended in 50 mM KPi buffer (pH 6.5). Using manual pH adjustment, the pH was maintained during the first 5 hours of the biotransformation process at pH 6.5. The bile salt concentrations are shown as a function of time in FIG. 16 . After 24 h, 92% of the product 12-keto-UDCA had formed.
  • One route for the synthesis of ursodesoxycholic acid comprises the chemical oxidation of cholic acid to dehydrocholic acid with subsequent asymmetric enzymatic reductions of the 3-carbonyl group to the 3 ⁇ -hydroxyl group and of the 7-carbonyl group to the 7 ⁇ -hydroxyl group followed by chemical removal of the 12-carbonyl group.
  • Suitable enzymes include formate dehydrogenases (FDH), glucose dehydrogenases (GDH), glucose-6-phosphate dehydrogenases (G6PDH), alcohol dehydrogenases (ADH) or phosphite dehydrogenases (PtDH).
  • FDH formate dehydrogenases
  • GDH glucose dehydrogenases
  • G6PDH glucose-6-phosphate dehydrogenases
  • ADH alcohol dehydrogenases
  • PtDH phosphite dehydrogenases
  • vectors were used according to the invention, which contain all three genes for 7 ⁇ -HSDH, 3 ⁇ -HSDH and FDH.
  • the vectors are constructed so that the expression level of the three genes in the designated host strain, especially whole-cell biocatalyst strains based on Escherichia coli BL21(DE3), are adapted in such a way that all three enzymes have enzyme activities that are as similar as possible.
  • the three genes required in the whole-cell reduction of dehydrocholic acid to 12-keto-ursodeoxycholic acid are located on one and the same vector. These are genes that code for the following enzymes: an NADPH-dependent 7 ⁇ -HSDH from Collinsella aerofaciens , an NADH-dependent 3 ⁇ -HSDH from Comamonas testosteroni , a mutated FDH from Mycobacterium vaccae that is both NAD- and NADP-dependent.
  • the expression levels of these three genes are balanced by the genetic construct, so that optimal whole-cell biocatalysis can be carried out.
  • FIG. 18 shows a schematic representation of the possible reaction pathways and reaction products.
  • the host strain used is a modified E. coli BL21(DE3) with the designation E. coli BL49.
  • the E. coli BL49 transformed with pF(G)r7(A)r3 bears the designation E. coli BL49 pF(G)r7(A)r3
  • the E. coli BL49 transformed with pF(G)r7(S)r3 bears the designation E. coli BL49 pF(G)r7(S)r3.
  • the biocatalysts were compared by whole-cell biotransformation at the 20-mL scale. For this, first a 5 mL overnight culture was grown in LB medium with 100 mg L-1 ampicillin. On the next day, 1 mL of this overnight culture was transferred into 200 mL of TB medium in a shaken flask with 100 mg of L-1 ampicillin. These cultures were cultured at 37° C. and 250 rpm in the shaking incubator, until an OD of 0.6-0.8 was reached. Then induction was carried out with 1 mM isopropyl- ⁇ -D-thiogalactopyranoside (IPTG). The subsequent expression phase was carried out for 21 h at 25° C. and 160 rpm. The cells were harvested by 10-minute centrifugation at 3220 g.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the reaction mixtures for the biotransformations contained 11.8 g L ⁇ 1 BTM whole-cell biocatalysts, 100 mM substrate (DHCA), 500 mM sodium formate and were suspended in 50 mM potassium formate buffer (pH 6.5). The process was carried out at the 20 mL scale as a batch process without pH monitoring.
  • the following table gives the results of the whole-cell biotransformation described above after a process time of 5 h.
  • the two new strains E. coli BL49 pF(G)r7(S)r3 and E. coli BL49 pF(G)r7(A)r3 are compared with a reference strain E. coli t BL49 pF(G)r7 pC3.
  • the following table shows proportions of bile salts in biotransformation batches after 5 h when using 100 mM substrate (DHCA).
  • the batches with the two new biocatalyst strains E. coli BL49 pF(G)r7(A)r3 and E. coli BL49 pF(G)r7(S)r3 are shown in comparison with the strain E. coli BL49 pF(G)r7 pC3 according to the prior art:
  • the biotransformation was set up in a 1 L bioreactor in the following reaction conditions: 70 mM DHCA, 17.7 g L ⁇ 1 BTM of the stored biocatalyst, 500 mM sodium formate, 26% (v/v) glycerol, 50 mM MgCl 2 , suspended in 50 mM KPi buffer (pH 6.5). Using pH adjustment, the pH was maintained at pH 6.5 throughout the biotransformation. The variation of the bile salt concentrations as a function of time is shown in FIG. 19 . After 21 h, 99.4% of product (12-keto-UDCA) had formed and only 0.6% of the by-product 3,12-diketo-UDCA was detectable.
  • a GDH from Bacillus subtilis that is both NAD- and NADP-dependent for the cofactor regeneration is expressed in a whole-cell biocatalyst (single-cell system) for the two-step reduction of DHCA to 12-keto-UDCA.
  • the nucleic acid sequence and the associated amino acid sequence of this GDH are given as SEQ ID NO:47 and SEQ ID NO:48 respectively.
  • the vectors used for this bear the designations p3T7(A)rG and p7(A)T3rG and are shown in FIG. 20 and FIG. 21 respectively.
  • the vectors according to the invention were transformed into Escherichia coli production strains. These strains represent the whole-cell biocatalyst.
  • the host strain used is a modified E. coli BL21(DE3) with the designation E. coli BL49 (identical to E. coli BL21(DE3) hdhA ⁇ KanR + ).
  • the host organism is not restricted to this host strain.
  • the E. coli BL49 transformed with p7(A)T3rG bears the designation E. coli BL49 p7(A)T3rG
  • the E. coli BL49 transformed with p3T7(A)rG bears the designation E. coli BL49 p3T7(A)rG.
  • the biocatalysts were compared by whole-cell biotransformation at the 20-mL scale. Cultivation and determination of the concentration of the substrate DHCA, of the intermediates 3,12-diketo-UDCA and 7,12-diketo-UDCA and of the product 12-keto-UDCA in the reaction mixture were carried out as described in reaction example 11.1.
  • the reaction mixtures for the biotransformations contained 11.8 g/L BTM whole-cell biocatalysts, 100 mM substrate (DHCA), 500 mM glucose and were suspended in 50 mM potassium formate buffer (pH 7.3). The process took place at the 20 mL scale as a batch process without pH monitoring. Perforation of the biocatalysts was not necessary.
  • the following table shows the results of the whole-cell biotransformation described above after a process time of 24 h, showing the results of the whole-cell biotransformation with the two strains E. coli BL49 p7(A)T3rG and E. coli BL49 p3T7(A)rG.
  • the strain E. coli BL49 p7(A)T3rG was cultured in the 7 L bioreactor at a working volume of 4 L in the defined minimal medium. After a brief initial phase at 37° C. there was a substrate-limited exponential growth phase at 30° C. On reaching an optical density of 25-30, protein expression of the cells was induced by adding 0.5 mM IPTG. Expression was then carried out for 24 h at 20° C.
  • the cells (46.7 g/L BTM) were harvested by centrifugation, resuspended in potassium phosphate buffer (pH 6.5) and stored according to the standard protocol at ⁇ 20° C. At the time of harvesting, the enzyme activities were: 16.4 U/mL 7 ⁇ -HSDH, 3.6 U/mL 3 ⁇ -HSDH, 44.9 U/mL GDH (NADP), 18.1 U/mL GDH (NAD).
  • Biotransformation was set up at the 20-mL scale in the following reaction conditions: 100 mM DHCA, 17.7 g/L BTM of the stored biocatalyst, 500 mM glucose, 10 mM MgCl 2 , suspended in 50 mM KPi buffer (pH 7). Using manual pH adjustment, the pH was maintained throughout the biotransformation at pH 7. The reactions were carried out either without cofactor addition or with addition of 0.1 mM NAD. The variation of the bile salt concentrations as a function of time is shown in FIG. 22 . After 2 h, with and without addition of NAD, in each case ⁇ 98% of product (12-keto-UDCA) was formed.
  • two whole-cell biocatalysts were used instead of one whole-cell biocatalyst.
  • an NADP-specific FDH from Mycobacterium vaccae and an NADPH-specific 7 ⁇ -HSDH from Collinsella aerofaciens were expressed in one of these biocatalysts, whereas an NAD-dependent FDH from Mycobacterium vaccae and an NADH-dependent 3 ⁇ -HSDH from Comamonas testosteroni were expressed in the other biocatalyst.
  • the vector pF(G)r7(A) which comprises genes for an NADP-specific FDH from Mycobacterium vaccae and for an NADPH-specific 7 ⁇ -HSDH from Collinsella aerofaciens
  • the vector pFr3 which comprises genes for an NAD-dependent FDH from Mycobacterium vaccae and for an NADH-dependent 3 ⁇ -HSDH from Comamonas testosteroni .
  • the vectors pF(G)r7(A) and pFr3 are shown in FIG. 23 a and b.
  • FIG. 24 shows a reaction scheme with possible routes and products.
  • the invention is not restricted to these two stated vectors, but can comprise all conceivable vectors that comprise genes for a 7 ⁇ -HSDH and a suitable cofactor regeneration enzyme in combination with vectors that comprise genes for a 3 ⁇ -HSDH and a suitable cofactor regeneration enzyme.
  • This example shows that the rate of the individual reaction steps can be influenced by adjusting the proportions of the biocatalysts used.

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US10954494B2 (en) 2021-03-23
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