PROCESS FOR THE ENANTIOSELECTIVE ENZYMATIC REDUCTION OF SECODIONE DERIVATIVES

Abstract

a process for the enantioselective enzymatic reduction of secodione derivatives. The secodione derivative is reduced with an oxidoreductase/dehydrogenase in the presence of NADH or NADPH as a cofactor. The secodione derivative is used in the reaction batch at a concentration of ≧10 g/l and the oxidized cofactor NAD or NADP formed by the oxidoreductase/dehydrogenase is regenerated continuously.

Description

[0001]The present invention relates to a process for the enantioselective enzymatic reduction of secodione derivatives of general formula I, wherein the secodione derivative is reduced with an oxidoreductase/dehydrogenase in the presence of NADH or NADPH as a cofactor.

[0002]The industrial preparation of steroid hormones occurs in two ways which are independent of each other, namely, on the one hand, starting out from naturally occurring steriod compounds from plant sources and, on the other hand, in a totally synthetic manner via an enantioselective synthesis from prochiral precursors. Among those two ways, the steroid total synthesis is increasingly gaining in importance, particularly since it also allows the introduction of structural elements which are not contained in naturally occurring steriods.

[0003]Key components of the total synthesis of enantiomerically pure steriods are thereby compounds of general formula I, which are also referred to as secosteroids, 8,14-seco-gona-tetraene-14,17-diones or secodiones. Specific representatives of this group are, for example, the compounds methyl secodione (Formula II, 13-methyl-3-methoxy-8,14-seco-gona-1,3,5(10),9(11)-tetraene-14,17-dione) and ethyl secodione (Formula III, 13-ethyl-3-methoxy-8,14-seco-gona-1,3,5(10),9(11)-tetraene-14,17-dione), from which, for example, the pharmacologically active compounds ethinyl estradiol (Formula IV) and norgestrel (Formula V) can be produced.

##STR00001##

[0004]A key step in the preparation of enantiomerically pure steroid compounds is the conversion of the compound of formula I (e.g., II and III) into an optically active compound with a preformed asymmetric C-13 by enantioselective reduction of one of the keto groups to the hydroxy group. The resulting optically active hydroxy secosteroid compounds (secoles, Formulae VI to IX) can subsequently be processed further into chiral steroid compounds by cyclization, while chirality is maintained.

[0005]By enantioselective reduction of a keto group of the compound of formula I, four optically active compounds can, in theory, be formed (Formulae VI to IX).

TABLE-US-00001 ##STR00002## Formula VI (17-beta-OH) ##STR00003## Formula VII (17-alpha-OH) ##STR00004## Formula VIII (14-beta-OH) ##STR00005## Formula IX (14-alpha-OH)

[0006]Compounds of formula VI, in which the hydroxy group exhibits the beta-configuration at position 17, are thereby of particular economic interest, since they result in derivatives of the natural estrone. Such compounds are also referred to as 17-beta-hydroxy secosteroids.

[0007]The stereoselective reduction of secodione derivatives of general formula I with the aid of different microorganisms was examined particularly thoroughly in the 60ies and 70ies. In doing so, it could be shown that different yeast strains of the genera Candida, Debaryomyces, Kloeckera, Pichia, Cryptococcus, Rhodotorula, Torulopsis and Hansenula are capable of reducing secodiones to various hydroxy compounds (U.S. Pat. No. 3,616,226, U.S. Pat. No. 1,252,524, U.S. Pat. No. 3,616,225).

[0008]In particular, yeasts of the genus Saccharomyces such as, e.g., S. uvarum can be used advantageously for preparing, for example, the respective 17-beta-hydroxy secosteroids (Kosmol et al; Liebigs Ann. Chem. 701, 199 (1967)). Other yeast strains such as, e.g., Saccharomyces drosophilarum reduce secodione preferably to the corresponding 14-alpha-hydroxy secosteroid (Acta microbiol. Acad. Sci. hung. 22, 463-471 (1975)). Furthermore, the formation of 14-alpha-hydroxy secosteroid is also described by the reduction of secodione by means of Bacillus thuringiensis (Kosmol et al.; Liebigs Ann. Chem. 701, 199 (1967)).

[0009]Gestagen and estrogen agents are widely used all over the world as contraceptives and in hormone replacement therapy. Most syntheses of estrogen and gestagen derivatives have to date been based on the above-described reaction principle, the key step of which is the enantioselective reduction of secodiones to the corresponding 17-beta-hydroxy secosteroids.

[0010]In doing so, the stereoselective reduction of secodione derivatives has to date been performed as a whole-cell biotransformation using different yeast strains of the genus Pichia or Saccharomyces. However, those processes have the disadvantage that only very low substrate concentrations of far below 1% (normally from 1 to 5 g/l) are feasible (U.S. Pat. No. 3,697,379; Current Science, Feb. 5 (1984), Vol 53. No. 3, p. 124; Indian Journal of Experimental Biology, Vol. 27, August 1989, p. 742-743). Thus, in particular the reprocessing and isolation of the reaction product from large volumes as well as the separation of large amounts of biomass turn out to be very complex. To the inventors' knowledge, the enzymes involved in the reduction have so far not been isolated, identified and described. Likewise, DNA sequences which code for oxidoreductases by means of which the reduction of secodione derivatives can be achieved have not yet been identified.

[0011]Thus, it is the object of the invention to provide a process by means of which secodione derivatives of general formula I, particularly those of formulae II and III, can be reduced enantioselectively. In this way, among other things, also the production of the corresponding 17-beta-hydroxy secosteroids should be rendered feasible.

[0012]In a first aspect, said object is achieved according to the invention by a process for the enantioselective enzymatic reduction of secodione derivatives of general formula I,

##STR00006##

wherein the ring structures comprise no, one or several heteroatoms,R₁ is hydrogen or a C₁-C₄ alkyl group,R₂ is hydrogen, a C₁-C₈ alkyl group or a protective group for OH known in prior art, such as an ester,R₃ is hydrogen, a methyl group or a halide,the structural element

##STR00007##

represents a benzene ring or a C₆ ring having 0, 1 or 2 C--C double bonds,a double bond is optionally included at positions 6/7 or 7/8, andthe carbon at positions 1, 2, 4, 5, 6, 7, 8, 9, 11, 12 and 16 is independently substituted with hydrogen, a C₁-C₄ alkyl group, a halide or a phenyl group,wherein the secodione derivative is reduced with an oxidoreductase/dehydrogenase in the presence of NADH or NADPH as a cofactor,which process is characterized in that the secodione derivative is used in the reaction batch at a concentration of ≧10 g/l and the oxidized cofactor NAD or NADP formed by the oxidoreductase/dehydrogenase is regenerated continuously.

[0013]This process represents a significant improvement of the enantioselective enzymatic reduction of secodione derivatives over the prior art. The process according to the invention allows the reduction of secodione derivatives to the different corresponding hydroxy secosteroids with free enzymes at concentration ranges far exceeding those described in the prior art.

[0014]In a second aspect, the above-mentioned object is achieved according to the invention by a process for the enantioselective enzymatic reduction of secodione derivatives of general formula I, wherein the secodione derivative is reduced with an oxidoreductase/dehydrogenase in the presence of NADH or NADPH as a cofactor, which process is characterized in that the oxidoreductase/dehydrogenase

a) comprises an amino acid sequence in which at least 50% of the amino acids are identical to those of amino acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5,b) is encoded by the nucleic acid sequence SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, orc) is encoded by a nucleic acid sequence which hybridizes to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 under stringent conditions.

[0015]The inventors have identified oxidoreductases which are capable of reducing secodione derivatives to hydroxy secosteroids and which can be produced recombinantly on an industrial scale. Significantly higher substrate concentrations can be achieved by the process according to the invention than with the currently used whole-cell processes.

[0016]In the process according to the invention, the oxidoreductase having the sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 or a polypeptide derivable from those polypeptides, respectively, can be used either in a completely purified state, in a partially purified state or as cells containing the polypeptide SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. Thereby, the cells used can be provided in a native, permeabilized or lysed state. Preferably, the oxidoreductases and derivatives derivable therefrom, respectively, are overexpressed in a suitable host organism such as, e.g., Escherichia coli, and the recombinant polypeptide is used for the reduction of secodione derivatives of general formula I.

[0017]A DNA sequence SEQ ID NO:6 which codes for a polypeptide with SEQ ID NO:1 is obtainable, for example, from the genome of the organism Chloroflexus aurantiacus DSM 635.

[0018]A DNA sequence SEQ ID NO:7 which codes for a polypeptide with SEQ ID NO:2 is obtainable, for example, from the genome of the organism Rubrobacter xylanophilus DSM 9941.

[0019]A DNA sequence SEQ ID NO:8 which codes for a polypeptide with SEQ ID NO:3 is obtainable from a yeast Candida magnoliae CBS 6396.

[0020]Oxidoreductases of SEQ ID NO:4 and SEQ ID NO:5 are obtainable, for example, from Candida magnoliae DSMZ 70638 by homology screening.

[0021]A nucleic acid sequence which hybridizes, for example, to SEQ ID NO:6 under stringent conditions is understood to be a polynucleotide which can be identified via the colony hybridization method, the plaque hybridization method, the Southern hybridization method or comparable methods, using SEQ ID NO:6 or partial sequences of SEQ ID NO:6 as a DNA probe. For this purpose, the polynucleotide immobilized on a filter is hybridized, for example, to SEQ ID NO:6 in a 0.7-1 M NaCl solution at 60° C. Hybridization is carried out as described, e.g., in Molecular Cloning, A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, 1989) or in similar publications. Subsequently, the filter is washed with a 0.1 to 2-fold SSC solution at 65° C., wherein a 1-fold SSC solution is understood to be a mixture consisting of 150 mM NaCl and 15 mM sodium citrate.

[0022]A polynucleotide which hybridizes to the polynucleotides SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 from the sequence list under the above-mentioned stringent conditions should exhibit at least 60% sequence identity to the polynucleotide sequences SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, better an identity of at least 80%, even better an identity of 95%.

[0023]In a further aspect, the above-mentioned object is achieved according to the invention by a process for the enantioselective enzymatic reduction of secodione derivatives of general formula I, wherein the secodione derivative is reduced with an oxidoreductase/dehydrogenase in the presence of NADH or NADPH as a cofactor, which process is characterized in that the oxidoreductase/dehydrogenase has a length of from 230 to 260 amino acids and comprises one or several of the partial sequences selected from the group consisting of [sequences SEQ ID NO:18 to SEQ ID NO:42]

nalvtgasrgig, nalvtggsrgig, nalitggsrgig, nalitgasrgig, nalitggsrgmg, halvtgasrgig,gysvtla, gynvtla, gysvtlv, gynvtlv,fkgaplpa, fkaaplpa,fvsnag, ffsnag, fvcnag, fvanag,spialtkal, spvaltkti, spialtktl, spvamtkal, sqialtkal,avysask, avysatk,pikgwi and pisgwi.

[0024]In the processes according to the invention, NADH or NADPH is used as the cofactor. By the term "NADP", nicotinamide adenine dinucleotide phosphate is understood, by "NADPH", reduced nicotinamide adenine dinucleotide phosphate is understood. The term "NAD" means nicotinamide adenine dinucleotide, the term "NADH" means reduced nicotinamide adenine dinucleotide.

[0025]According to a preferred embodiment of the process in which the secodione derivative is used in the reaction batch at a concentration of ≧10 g/l and the oxidized cofactor NAD or NADP formed by the oxidoreductase/dehydrogenase is regenerated continuously, the oxidoreductase/dehydrogenase

a) comprises an amino acid sequence in which at least 50% of the amino acids are identical to those of amino acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5,b) the oxidoreductase/dehydrogenase is encoded by the nucleic acid sequence SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, orc) the oxidoreductase/dehydrogenase is encoded by a nucleic acid sequence which hybridizes to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 under stringent conditions.

[0026]According to another preferred embodiment of the process in which the secodione derivative is used in the reaction batch at a concentration of ≧10 g/l and the oxidized cofactor NAD or NADP formed by the oxidoreductase/dehydrogenase is regenerated continuously, the oxidoreductase/dehydrogenase has a length of from 230 to 260 amino acids and comprises one or several of the partial sequences selected from the group consisting of [sequences SEQ ID NO:18 to SEQ ID NO:42] nalvtgasrgig, nalvtggsrgig, nalitggsrgig, nalitgasrgig, nalitggsrgmg, halvtgasrgig, gysvtla, gynvtla, gysvtlv, gynvtlv, fkgaplpa, fkaaplpa, fvsnag, ffsnag, fvcnag, fvanag, spialtkal, spvaltkti, spialtktl, spvamtkal, sqialtkal, avysask, avysatk, pikgwi and pisgwi.

[0027]In the processes according to the invention, which refer to the second and third aspects of the invention, the oxidized cofactor NAD or NADP formed by the oxidoreductase/dehydrogenase is preferably regenerated continuously.

[0028]According to a preferred embodiment of all processes according to the invention, the oxidized cofactor NAD or NADP is regenerated by oxidation of an alcohol.

[0029]In doing so, primary and secondary alcohols such as ethanol, 2-propanol, 2-butanol, 2-pentanol, 3-pentanol, 4-methyl-2-pentanol, 2-hexanol, 2-heptanol, 2-octanol or cyclohexanol are preferably used as cosubstrates. The proportion of the cosubstrate for the regeneration may range from 5 to 95% by volume, based on the total volume.

[0030]A secondary alcohol having the general formula R_XRYCHOH is preferably used for cofactor regeneration, wherein R_X and R_Y independently of each other are hydrogen, a branched or unbranched C₁-C₈ alkyl group and C_total≧3.

[0031]According to another preferred embodiment of the processes according to the invention, an oxidoreductase/dehydrogenase is additionally added for the regeneration of the cofactor.

[0032]Suitable NADH-dependent alcohol dehydrogenases are, for example, obtainable from baker's yeast, from Candida parapsilosis (CPCR) (U.S. Pat. No. 5,523,223 and U.S. Pat. No. 5,763,236, Enzyme Microb. Technol., 1993, 15(11):950-8), Pichia capsulata (DE 10327454.4), from Rhodococcus erythropolis (RECR) (U.S. Pat. No. 5,523,223), Norcardia fusca (Biosci. Biotechnol. Biochem., 63(10), 1999, p. 1721-1729; Appl. Microbiol. Biotechnol, 2003, 62(4):380-6; Epub 2003, Apr. 26) or Rhodococcus ruber (J. Org. Chem., 2003, 68(2):402-6). Suitable cosubstrates for those alcohol dehydrogenases are, for example, the already mentioned secondary alcohols such as 2-propanol (isopropanol), 2-butanol, 2-pentanol, 4-methyl-2-pentanol, 2-octanol or cyclohexanol.

[0033]Suitable secondary alcohol dehydrogenases for the regeneration of NADPH are, for example, those as described above and isolated from organisms of the order of Lactobacillales, e.g., Lactobacillus kefir (U.S. Pat. No. 5,200,335), Lactobacillus brevis (DE 19610984 A1; Acta Crystallogr. D. Biol. Crystallogr. 2000 December; 56 Pt 12:1696-8), Lactobacillus minor (DE 10119274), Leuconostoc carnosum (A 1261/2005, K1. C12N) or, as described, those from Thermoanerobium brockii, Thermoanerobium ethanolicus or Clostridium beijerinckii.

[0034]However, other enzymatic systems can, in principle, also be used for cofactor regeneration. For example, cofactor regeneration can be effected using NAD- or NADP-dependent formate dehydrogenase (Tishkov et al., J. Biotechnol. Bioeng. [1999] 64, 187-193, Pilot-scale production and isolation of recombinant NAD and NADP specific formate dehydrogenase). Suitable cosubstrates of formate dehydrogenase are, for example, salts of formic acid such as ammonium formate, sodium formate or calcium formate.

[0035]The TTN (total turn over number=mol of reduced secodione compound/mol of cofactor used) achieved in the processes according to the invention normally ranges from 10² to 10⁵, preferably, however, it is ≧10³.

[0036]According to a preferred embodiment, the processes according to the invention are carried out in an aqueous organic two-phase system.

[0037]Accordingly, the conversion of the secodione derivative occurs in a two-phase system containing, for example, a 2-alcohol for cofactor regeneration, an oxidoreductase, water, cofactor and the secodione compound. However, additional organic solvents which are not involved in the cofactor regeneration, i.e., do not contain any oxidizable hydroxy groups, can also be included. Diethyl ether, tertiary butyl methyl ether, diisopropyl ether, dibutyl ether, ethyl acetate, butyl acetate, heptane, hexane, toluene, dichloromethane, cyclohexane or mixtures thereof are preferably used as additional organic solvents.

[0038]Thereby, the amount of non-water-miscible organic components of the two-phase system may range from 10% to 90%, preferably from 20% to 80%, based on the total volume of the reaction batch. The aqueous amount may range from 90% to 10%, preferably from 80% to 20%, based on the total volume of the reaction batch.

[0039]A buffer can also be added to the water, for example, a potassium phosphate, tris/HCl, glycine or triethanolamine buffer, having a pH value of from 5 to 10, preferably from 6 to 9. In addition, the buffer can comprise ions for stabilizing or activating both enzymes, for example, magnesium ions or zinc ions.

[0040]Moreover, further additives for stabilizing the enzymes used can be used in the processes according to the invention, for example, glycerol, sorbitol, 1,4-DL-dithiothreitol (DTT) or dimethyl sulfoxide (DMSO).

[0041]The concentration of the cofactor NAD(P)H, based on the aqueous phase, ranges from 0.001 mM to 10 mM, in particular from 0.01 mM to 1.0 mM. Depending on the specific properties of the enzymes used, the temperature can be from 10° C. to 70° C., preferably from 20° C. to 35° C.

[0042]Normally, the secodione derivatives to be reduced are poorly soluble in water. Therefore, the substrate can be provided in a completely or also incompletely dissolved state during the reaction. If the substrate is not dissolved completely in the reaction mixture, a portion of the substrate is present in a solid form and can thus form a third solid phase. The reaction mixture may also temporarily form an emulsion during the conversion.

[0043]In the processes according to the invention, the secodione derivative of general formula I is used in the reaction batch preferably in an amount of from 10 g/l to 500 g/l, preferably from 25 g/l to 300 g/l, particularly preferably from 50 g/l to 200 g/l, based on the total volume.

[0044]Preferred embodiments of the invention are furthermore characterized in that 13-ethyl-3-methoxy-8,14-seco-gona-1,3,5 (10),9(11)-tetraene-14,17-dione (ethyl secodione--Formula III) or 13-methyl-3-methoxy-8,14-seco-gona-1,3,5(10),9(11)-tetraene-14,17-dione (methyl secodione--Formula II) is used as the secodione derivative.

[0045]The processes according to the invention are carried out, for example, in a reaction vessel made of glass or metal. For this purpose, the components are transferred individually into the reaction vessel and stirred under an atmosphere of, e.g., nitrogen or air. The reaction time ranges from one hour to 7 days, in particular from 2 hours to 48 hours, depending on the secodione compound and the oxidoreductase used. During that time, the secodione compound is reduced to the corresponding hydroxy secosteroid compound by at least 50%.

[0046]Below, the present invention is illustrated in more detail by way of examples.

EXAMPLE 1

Cloning of an Oxidoreductase from Chloroflexus auratiacus DSM 635

[0047]A) Cultivation of Chloroflexus auratiacus DSM 635

[0048]Cells of Chloroflexus auratiacus DSM 635 were cultivated in a bacterial incubator in the following medium (pH 8.2) at 48° C. under light: 0.1% yeast extract, 0.1% glycyl glycine, 0.01% Na₂HPO₄×2H₂O, 0.01% MgSO₄×7H₂O, 0.01% KNO₃, 0.05% NaNO₃, 0.01% NaCl, 0.005% CaCl₂×2H₂O, 5 ml of a 0.01% Fe(III) citrate solution, 1 ml of trace element solution SL-6 [500 μl/l H₂SO₄, 2.28 g/l MnSO₄×H₂O, 500 mg/l ZnSO₄×7H₂O, 500 mg H₃BO₃, 25 mg/l CuSO₄×5H₂O, 25 mg/l Na₂MoO₄×2H₂O, 45 mg/l CoCl₂×6H₂O]. On day 12 of the cultivation, cells were separated from the culture medium by centrifugation and stored at -80° C.

B) Amplification of the Gene Coding for Selective Oxidoreductase

[0049]Genomic DNA was extracted according to the method described in "Molecular Cloning" by Manniatis & Sambrook. The resulting nucleic acid served as a template for the polymerase chain reaction (PCR) involving specific primers which were derived from the gene sequence published under number 76258197 in the NCBI database. In doing so, the primers were provided in a 5'-terminal position with restriction sites for the endonucleases Nde I and Hind III or Sph I, respectively (SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13), for subsequent cloning into an expression vector.

[0050]Amplification was carried out in a PCR buffer [10 mM Tris-HCl, (pH 8.0); 50 mM KCl; 10 mM MgSO₄; 1 mM dNTP Mix; in each case 20 pMol of primer and 2.5 U of Platinum Pfx DNA Polymerase (Invitrogen)] with 500 ng of genomic DNA and the following temperature cycles:

Cycle 1: 94° C., 2 min

Cycle 2×30: 94° C., 30 sec

[0051]56° C., 30 sec [0052]68° C., 60 sec

Cycle 3: 68° C., 7 min

[0052] [0053]4° C., ∞

[0054]The resulting PCR product with a size of about 750 by was restricted after purification over a 1% agarose gel with the aid of the endonucleases Nde I and Hind III or endonucleases Sph I and Hind III, respectively, and was ligated into the backbone of the pET21a vector (Novagen) or of the pQE70 vector (Qiagen), respectively, which backbone had been treated with the same endonucleases. After transforming 2 μl of the ligation batch into E. coli Top 10 F cells (Invitrogen), plasmid DNAs of ampicillin (or kanamycin)-resistant colonies were tested for the presence of an insert having a size of 750 by means of a restriction analysis with the endonucleases Nde I and Hind III or endonucleases Sph I and Hind III, respectively. Plasmid preparations from the clones which were positive for the fragment were subjected to a sequence analysis and subsequently transformed into Escherichia coli BL21 Star (Invitrogen) and E. coli RB791 (genetic stock, Yale), respectively.

EXAMPLE 2

Expression of Recombinant Chloroflexus Oxidoreductase in E. coli

[0055]The Escherichia coli strains BL21 Star (Invitrogen, Karlsruhe, Germany) and RB791 (E. coli genetic stock, Yale, USA), respectively, transformed with the expression construct were cultivated in 200 ml LB-medium (1% tryptone, 0.5% yeast extract, 1% NaCl) with ampicillin (50 μg/ml) or carbenicillin (50 μg/ml), respectively, until an optical density (OD) of 0.5, measured at 550 nm, was reached. The expression of recombinant protein was induced by adding isopropylthiogalactoside (IPTG) at a concentration of 0.1 mM. After 8 hours or 16 hours of induction at 25° C. and 220 rpm, the cells were harvested and frozen at -20° C. For the activity test, 10 mg of cells were mixed with 500 μl of 100 mM TEA buffer pH 7.0 and 500 μl of glass beads and digested for 10 min using a globe mill. The lysate obtained was then used in a diluted state for the respective measurements. The activity test was made up as follows: 870 μl of 100 mM TEA buffer pH 7.0, 160 μg NADH, 10 μl of diluted cell lysate. The reaction was started by adding 100 μl of a 100 mM substrate solution to the reaction mixture.

[0056]For enzyme recovery in large amounts, 30 g of cells were resuspended in 150 ml of triethanolamine buffer (100 mM, pH 7, 2 mM MgCl₂, 10% glycerol) and digested using a high-pressure homogenizer. Subsequently, the enzyme solution was mixed with 150 ml glycerol and stored at -20° C.

EXAMPLE 3

Cultivation of Organisms and Screening after a Reductive Conversion of Ethyl Secodione (Formula III)

[0057]For screening, the yeast strains Pichia farinosa DSM 70362, Candida gropengiesseri MUCL 29836, Candida vaccinii CBS 7318, Pichia farinosa DSM 3316, Saccharomyces cerevisiae CBS 1508 and Candida magnoliae CBS 6396 were cultivated in the following medium: yeast extract (5), peptone (5) and glucose (20) (the numbers in brackets are, in each case, g/l). The medium was sterilized at 121° C. and the yeasts were cultivated at 25° C. on a shaker at 140 revolutions per minute without further pH-adjustment.

[0058]The reductive conversion of ethyl secodione of formula III to the corresponding hydroxy secosteroid compound was tested in the following whole-cell biotransformation batches: 400 mg of freshly harvested cells were shaken in a batch with 50 mg glucose, 10 mg ethyl secodione of formula III and 900 μl of 100 mM trieethanolamine buffer (TEA) pH 7.0 at 28° C. and 1400 rpm for 24 hours. Subsequently, the batches were extracted with 1 ml of dichloromethane, centrifuged, dried with nitrogen and, after having been absorbed in acetonitrile, added to the HPLC analysis.

[0059]The screening results are summarized in Table 1.

TABLE-US-00002 TABLE 1 Conversion of ethyl secodione after 24 hours with Wt strains Strain no. Microorganism Batch 24 h DSM 70362 Pichia farinosa 0.7% MUCL 29836 Candida gropengiesseri 0.2% CBS 7318 Candida vaccinii 3.2% DSM 3316 Pichia farinosa 15.8% CBS 1508 Saccharomyces cerevisiae 0.7% CBS 6396 Candida magnoliae 41%

[0060]Strain CBS 6396 displayed the highest conversion of ethyl secodione and was thus chosen as the starting organism for the preparation of a cDNA library.

EXAMPLE 4

Preparation of a cDNA Library from Candida magnoliae Cbs 6396 and Cloning of Oxidoreductase

[0061]A) Isolation (Total and mRNA) as Well as Preparation of the cDNA Library

[0062]600 mg of fresh cells were resuspended in 2.5 ml of ice-cold LETS buffer. 5 ml (about 20 g) of glass beads washed in nitric acid and equilibrated with 3 ml phenol (pH 7.0) were added to said cell suspension. The entire batch was then alternately treated by 30 sec of vortexing and 30 sec of cooling on ice, in total for 10 minutes. Subsequently, 5 ml of ice-cold LETS buffer was added, and this was again vigorously vortexed. Said cell suspension was centrifuged at 4° C. with 11000 g for 5 minutes. The aqueous phase was recovered and extracted twice with an equal volume of phenol:chloroform:isoamyl alcohol (24:24:1). This was subsequently followed by the extraction with chloroform. After the final extraction, the total RNA was precipitated at -20° C. for 4 h by adding 1/10 vol. of 5 M LiCl₂.

[0063]1 mg of total RNA thus obtained was used via Oligo-dT cellulose (NEB Biolabs) for the enrichment of the mRNA molecules. After the subsequent precipitation, 5 μg mRNA was used for the cDNA synthesis (pBluescript IIXR cDNA Library Construction kit, Stratagene). The library constructed according to the manufacturer's instructions was transformed into XL-10 Gold E. coli and screened for the activity of an ADH. A clone (cM4) was identified and isolated based on the decrease in absorbance with NADPH or NADH, respectively, as the cofactor and ethyl secodione (Formula III) as the substrate. The sequencing of the plasmid isolated from the clone with primer T7 and primer T3 resulted in an ORF of 789 bp. Said fragment coded for a fusion protein having a size of 262 amino acids and consisted of the a-fragment of the β-galactosidase and the sequence of a putative short-chain alcohol dehydrogenase.

B) Synthesis of a Full-Length Transcript Coding for a Short-Chain ADH from Candida magnoliae CBS 6396 by PCR

[0064]Specific primers were constructed for subsequent cloning of the full-length transcript into the appropriate expression systems. In doing so, a 5'-primer with a recognition sequence for Nde I and Sph I, respectively, and a 3'-primer with a recognition sequence for XhoI and Sad, respectively, were modified (SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17). Plasmid DNA isolated from the clone (cM4) of the expression library of Candida magnoliae served as a template for the polymerase chain reaction. Amplification was carried out in a PCR buffer [10 mM Tris-HCl (pH 8.0); 50 mM KCl; 10 mM MgSO₄; 1 mM dNTP Mix; in each case 20 pMol of primer and 2.5 U of Platinum Pfx DNA Polymerase (Invitrogen)] with 50 ng of template and the following temperature cycles:

Cycle 1: 94° C., 2 min

Cycle 2×30: 94° C., 15 sec

[0065]58° C., 30 sec [0066]68° C., 75 sec

Cycle 3: 68° C., 7 min

[0066] [0067]4° C., ∞

[0068]The resulting PCR product was restricted after purification over a 1% agarose gel with the aid of the endonucleases Nde I and Xho I or the endonucleases Sph I and Sac I, respectively, and was ligated into the backbone of the pET21a vector (Novagen) or of the pQME70 vector, respectively, which backbone had been treated with the same endonucleases. After transforming 2 μl of the ligation batch into E. coli Top 10 F cells (Invitrogen), plasmid DNAs of ampicillin (or kanamycin)-resistant colonies were tested for the presence of an insert having a size of 750 by means of a restriction analysis with the endonucleases Nde I and XhoI or the endonucleases Sph I and SacI, respectively. The expression constructs pET21-MgIV and pQME70-MgIV were sequenced. The gene from Candida magnoliae coding for a short-chain oxidoreductase had an open reading frame of a total of 729 by (contained in SEQ ID NO:8), which corresponded to a protein of 243 amino acids (SEQ ID NO:3).

EXAMPLE 5

Expression of Recombinant Oxidoreductase in E. coli Cells

[0069]Competent Escherichia coli StarBL21(De3) cells (Invitrogen) and RB791 cells (E. coli genetic stock, Yale, USA), respectively, were transformed with the expression constructs pET21-MgIV and pQME70-MgIV, respectively, coding for the oxidoreductase. The Escherichia coli colonies transformed with the expression constructs were then cultivated in 200 ml of LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl) with 50 μg/ml of ampicillin or 40 μg/ml of kanamycin, respectively, until an optical density of 0.5, measured at 550 nm, was reached. The expression of recombinant protein was induced by adding isopropylthiogalactoside (IPTG) at a concentration of 0.1 mM. After 16 hours of induction at 25° C. and 220 rpm, the cells were harvested and frozen at -20° C. For the activity test, 10 mg of cells were mixed with 500 μl of 100 mM TEA buffer pH 7.0, 1 mM MgCl₂ and 500 μl glass beads and digested for 10 min using a globe mill. The lysate obtained was then used in a diluted state for the respective measurements.

[0070]The activity test was made up as follows: 960 μl of 100 mM TEA buffer pH 7.0, 1 bmM MgCl₂, 160 μg NADPH, 10 μl of diluted cell lysate. The reaction was started by adding 10 μl of a 100 mM substrate solution in 70% methanol to the reaction mixture.

[0071]For enzyme recovery in large amounts, 30 g of cells were resuspended in 150 ml of triethanolamine buffer (100 mM, pH 7, 2 mM MgCl₂, 10% glycerol) and digested using a high-pressure homogenizer. Subsequently, the enzyme solution was mixed with 150 ml glycerol and stored at -20° C.

EXAMPLE 6

Reduction of Ethyl Secodione (Formula III) Via Oxidoreductase SEQ ID NO:1

[0072]For the reduction of ethyl secodione (Formula III), a mixture of 800 μl buffer (100 mM potassium phosphate, pH=7, 2 mM MgCl₂), 1.2 ml 2-propanol, 0.08 mg NAD, 100 mg ethyl secodione (Formula III) and 1 ml of the enzyme suspension oxidoreductase SEQ ID NO:1 (see Example 3) was incubated in a reaction vessel at room temperature for 24 h under constant thorough mixing. After 96 h, >90% of the ethyl secodione (Formula III) used had been reduced.

[0073]Upon completion of the reaction, the reaction mixture was reprocessed by extraction with dichloromethane, the organic phase containing the product was separated and the 17-beta-hydroxy compound (ethyl secol) was obtained by evaporating/distilling off the solvent.

[0074]The conversion of the ethyl secodione into ethyl secol was followed via HPLC. For this purpose, a separating column EC125/4 Nucleodur 100-5 C18ec (Machery-Nagel, Duren, Germany) with acetonitrile and water as solvents was used. For analytics, a linear gradient of the acetonitrile portion in the solvent from 30% to 70% was applied. Identification of the reaction products was performed by comparison with reference substances.

EXAMPLE 7

Reduction of Ethyl Secodione (Formula III) Via Oxidoreductase SEQ ID NO:2

[0075]For the reduction of ethyl secodione (Formula III), a mixture of 250 μl buffer (100 mM triethanolamine, pH=8, 2 mM MgCl₂), 250 μl 4-methyl-2-pentanol, 0.02 mg NAD, 25 mg ethyl secodione (Formula III) and 25 μl of the enzyme suspension oxidoreductase SEQ ID NO:2 (see Example 3) was incubated in a reaction vessel at room temperature for 96 h under constant thorough mixing. After 96 h, >30% of the ethyl secodione (Formula III) used had been reduced to the hydroxy compound.

[0076]Upon completion of the reaction, the reaction mixture was reprocessed by extraction with dichloromethane, the organic phase containing the product was separated and the 17-beta-hydroxy compound (ethyl secol) was obtained by evaporating/distilling off the solvent.

EXAMPLE 8

Reduction of Ethyl Secodione (Formula III) Via Oxidoreductase SEQ ID NO:3

[0077]For the reduction of ethyl secodione (Formula III), a mixture of 100 μl buffer (100 mM triethanolamine, pH=7, 2 mM MgCl₂), 400 μl 4-methyl-2-pentanol, 0.02 mg NADP, 25 mg ethyl secodione (Formula III) and 100 μl of the enzyme suspension oxidoreductase SEQ ID NO:3 (see Example 3) was incubated in a reaction vessel at room temperature for 72 h under constant thorough mixing. After 72 h, >95% of the ethyl secodione (Formula III) used had been reduced to the hydroxy compound.

EXAMPLE 9

Reduction of Ethyl Secodione (Formula III) Via Oxidoreductase SEQ ID NO:4

[0078]For the reduction of ethyl secodione (Formula III), a mixture of 200 μl buffer (100 mM triethanolamine, pH=9, 2 mM MgCl₂), 300 μl 2-heptanol, 0.025 mg NADP, 100 mg ethyl secodione (Formula III) and 50 μl of the enzyme suspension oxidoreductase SEQ ID NO:4 (see Example 3) was incubated in a reaction vessel at room temperature for 72 h under constant thorough mixing. After 72 h, >80% of the ethyl secodione (Formula III) used had been reduced to the hydroxy compound.

EXAMPLE 10

Reduction of Ethyl Secodione (Formula III) Via Oxidoreductase SEQ ID NO:5

[0079]For the reduction of ethyl secodione (Formula III), a mixture of 300 μl buffer (100 mM triethanolamine, pH=7, 2 mM MgCl₂), 1.2 ml 4-methyl-2-pentanol, 0.12 mg NADP, 150 mg ethyl secodione (Formula III) and 0.6 ml of the enzyme suspension oxidoreductase SEQ ID NO:5 (see Example 3) was incubated in a reaction vessel at room temperature for 72 h under constant thorough mixing. After 72 h, >90% of the ethyl secodione (Formula III) used had been reduced to the hydroxy compound.

Sequence CWU 1

421252PRTChloroflexus auratiacus DSM 635 1Met Glu Pro Pro Phe Ile Gly Lys Val Ala Leu Val Thr Gly Ala Ala1 5 10 15Ala Gly Ile Gly Arg Ala Ser Ala Leu Ala Phe Ala Arg Glu Gly Ala 20 25 30Lys Val Val Val Ala Asp Val Asn Val Glu Gly Gly Glu Glu Thr Ile 35 40 45Ala Leu Cys Arg Ala Leu Asn Thr Asp Ala Met Phe Val Arg Cys Asp 50 55 60Val Ser Gln Arg Asp Glu Val Glu Arg Leu Ile Ala Leu Ala Val Asp65 70 75 80Thr Phe Gly Arg Ile Asp Phe Ala His Asn Asn Ala Gly Ile Glu Gly 85 90 95Val Gln Ala Met Leu Ala Asp Tyr Pro Glu Glu Val Trp Asp Arg Val 100 105 110Ile Glu Ile Asn Leu Lys Gly Val Trp Leu Cys Met Lys Tyr Glu Ile 115 120 125Arg His Met Leu Lys Gln Gly Gly Gly Ala Ile Val Asn Thr Ser Ser 130 135 140Val Ala Gly Leu Ala Gly Ser Arg Gly Val Ser Ala Tyr Val Ala Ser145 150 155 160Lys His Gly Ile Val Gly Ile Thr Lys Ala Ala Ala Leu Glu Tyr Ala 165 170 175Arg Asn Gly Ile Arg Val Asn Ala Ile Cys Pro Gly Thr Ile His Thr 180 185 190Ala Met Ile Asp Arg Phe Thr Gln Gly Asp Pro Gln Leu Leu Ala Gln 195 200 205Phe Ala Glu Gly Glu Pro Ile Gly Arg Leu Gly Ser Pro Glu Glu Val 210 215 220Ala Asn Ala Val Ile Trp Leu Cys Ser Asp Lys Ala Ser Phe Val Thr225 230 235 240Gly Ala Thr Leu Ala Val Asp Gly Gly Arg Leu Ala245 2502249PRTRubrobacter xylanophilus DSM 9941 2Met Leu Glu Gly Lys Val Ala Val Ile Thr Gly Ala Gly Ser Gly Ile1 5 10 15Gly Arg Ala Thr Ala Leu Lys Phe Ala Arg Glu Gly Ala Arg Val Val 20 25 30Ala Ala Glu Leu Asp Glu Arg Gly Gly Glu Gly Val Val Arg Glu Val 35 40 45Arg Ser Leu Gly Gly Glu Ala Val Phe Val Arg Thr Asp Val Ser Glu 50 55 60Phe Ala Gln Val Glu Asp Ala Val Glu Arg Ala Val Gly Glu Tyr Gly65 70 75 80Thr Leu Asp Val Met Phe Asn Asn Ala Gly Ile Gly His Tyr Ala Pro 85 90 95Leu Leu Glu His Glu Pro Glu His Tyr Asp Arg Val Val Arg Val Asn 100 105 110Gln Tyr Gly Val Tyr Tyr Gly Ile Leu Ala Ala Gly Arg Lys Met Val 115 120 125Ala Leu Lys Asn Pro Gly Leu Ile Ile Asn Thr Ala Ser Val Tyr Ala 130 135 140Phe Leu Ala Ser Pro Gly Val Ile Gly Tyr His Ala Ala Lys Gly Ala145 150 155 160Val Lys Met Met Thr Gln Ala Ala Ala Leu Glu Leu Ala Pro His Gly 165 170 175Ile Arg Val Val Ala Ile Ala Pro Gly Gly Val Asp Thr Pro Ile Ile 180 185 190Gln Gly Tyr Lys Asp Met Gly Leu Gly Glu Arg Leu Ala Arg Gly Gln 195 200 205Met Arg Arg Arg Leu Gln Thr Pro Glu Gln Ile Ala Gly Ala Val Ala 210 215 220Leu Leu Ala Thr Asp Glu Ala Asp Ala Ile Asn Gly Ser Val Val Met225 230 235 240Thr Asp Asp Gly Tyr Ala Glu Phe Lys 2453243PRTCandida magnoliae CBS 6396 3Met Ser Ala Thr Ser Asn Ala Leu Ile Thr Gly Ala Ser Arg Gly Met1 5 10 15Gly Glu Ala Thr Ala Ile Lys Leu Ala Leu Glu Gly Tyr Ser Val Thr 20 25 30Leu Ala Ser Arg Gly Ile Glu Gln Leu Asn Ala Ile Lys Glu Lys Leu 35 40 45Pro Ile Val Lys Lys Gly Gln Gln His Tyr Val Trp Gln Leu Asp Leu 50 55 60Ser Asp Ile Glu Ala Ala Ser Thr Phe Lys Gly Ala Pro Leu Pro Ala65 70 75 80Ser Ser Tyr Asp Val Phe Phe Ser Asn Ala Gly Val Val Asp Phe Ala 85 90 95Pro Phe Ala Asp Gln Ser Glu Thr Ala Gln Lys Asp Leu Phe Thr Val 100 105 110Asn Leu Leu Ser Pro Val Ala Leu Thr Lys Thr Ile Val Lys Ala Ile 115 120 125Ala Asp Lys Pro Arg Glu Thr Pro Ala His Ile Ile Phe Thr Ser Ser 130 135 140Ile Val Gly Ile Arg Gly Val Pro Asn Val Ala Val Tyr Ser Ala Thr145 150 155 160Lys Gly Ala Ile Asp Ser Phe Ala Arg Ser Leu Ala Arg Glu Phe Gly 165 170 175Pro Lys Asn Ile His Val Asn Cys Val Asn Pro Gly Thr Thr Arg Thr 180 185 190Glu Met Thr Lys Gly Val Asp Leu Ala Ala Phe Gly Asp Val Pro Ile 195 200 205Lys Gly Trp Ile Glu Val Asp Ala Ile Ala Asp Ala Val Leu Phe Leu 210 215 220Ile Lys Ser Lys Asn Ile Thr Gly Gln Ser Leu Val Val Asp Asn Gly225 230 235 240Phe Gly Val4241PRTCandida magnoliae DSM 70638 4Met Thr Ser Thr Pro Asn Ala Leu Ile Thr Gly Gly Ser Arg Gly Ile1 5 10 15Gly Ala Ser Ala Ala Ile Lys Leu Ala Gln Glu Gly Tyr Ser Val Thr 20 25 30Leu Ala Ser Arg Asp Leu Glu Lys Leu Thr Glu Val Lys Asp Lys Leu 35 40 45Pro Ile Val Arg Gly Gly Gln Lys His Tyr Val Trp Gln Leu Asp Leu 50 55 60Ala Asp Val Glu Ala Ala Ser Ser Phe Lys Ala Ala Pro Leu Pro Ala65 70 75 80Ser Ser Tyr Asp Leu Phe Val Ser Asn Ala Gly Ile Ala Gln Phe Ser 85 90 95Pro Thr Ala Glu His Thr Asn Ser Glu Trp Leu Asn Ile Met Thr Ile 100 105 110Asn Leu Val Ser Pro Ile Ala Leu Thr Lys Ala Leu Leu Gln Ala Val 115 120 125Ser Gly Arg Ser Ser Glu Asn Pro Phe Gln Ile Val Phe Ile Ser Ser 130 135 140Val Ala Ala Leu Arg Gly Val Ala Gln Thr Ala Val Tyr Ser Ala Ser145 150 155 160Lys Ala Gly Thr Asp Gly Phe Ala Arg Ser Leu Ala Arg Glu Leu Gly 165 170 175Pro Gln Gly Val His Val Asn Val Val Asn Pro Gly Trp Thr Lys Thr 180 185 190Asp Met Thr Glu Gly Val Glu Thr Pro Lys Asp Met Pro Ile Lys Gly 195 200 205Trp Ile Gln Pro Glu Ala Ile Ala Asp Ala Val Val Phe Leu Ala Arg 210 215 220Ser Lys Asn Ile Thr Gly Ala Asn Ile Val Val Asp Asn Gly Phe Ser225 230 235 240Thr5241PRTCandida magnoliae DSM 70638 5Met Thr Thr Thr Ser Asn Ala Leu Val Thr Gly Gly Ser Arg Gly Ile1 5 10 15Gly Ala Ala Ser Ala Ile Lys Leu Ala Gln Glu Gly Tyr Asn Val Thr 20 25 30Leu Ala Ser Arg Ser Val Asp Lys Leu Asn Glu Val Lys Ala Lys Leu 35 40 45Pro Ile Val Gln Asp Gly Gln Lys His Tyr Ile Trp Glu Leu Asp Leu 50 55 60Ala Asp Val Glu Ala Ala Ser Ser Phe Lys Gly Ala Pro Leu Pro Ala65 70 75 80Arg Ser Tyr Asp Val Phe Val Ser Asn Ala Gly Val Ala Ala Phe Ser 85 90 95Pro Thr Ala Asp His Asp Asp Lys Glu Trp Gln Asn Leu Leu Ala Val 100 105 110Asn Leu Ser Ser Pro Ile Ala Leu Thr Lys Ala Leu Leu Lys Asp Val 115 120 125Ser Glu Arg Pro Val Asp Lys Pro Leu Gln Ile Ile Tyr Ile Ser Ser 130 135 140Val Ala Gly Leu His Gly Ala Ala Gln Val Ala Val Tyr Ser Ala Ser145 150 155 160Lys Ala Gly Leu Asp Gly Phe Met Arg Ser Val Ala Arg Glu Val Gly 165 170 175Pro Lys Gly Ile His Val Asn Ser Ile Asn Pro Gly Tyr Thr Lys Thr 180 185 190Glu Met Thr Ala Gly Ile Glu Ala Leu Pro Asp Leu Pro Ile Lys Gly 195 200 205Trp Ile Glu Pro Glu Ala Ile Ala Asp Ala Val Leu Phe Leu Ala Lys 210 215 220Ser Lys Asn Ile Thr Gly Thr Asn Ile Val Val Asp Asn Gly Leu Ile225 230 235 240Ala6759DNAChloroflexus aurantiacus DSM 635 6atggagccac ctttcattgg gaaggttgcg ctggtcaccg gcgcagcagc cggtattggt 60cgtgcttcag cactggcgtt tgcccgtgag ggtgccaagg ttgtcgttgc tgatgtgaat 120gtcgagggcg gggaagagac gattgcgctg tgtcgggctt tgaataccga tgcaatgttc 180gtgcgttgtg atgtttcgca acgcgatgaa gtggagcgat taattgctct ggcagttgac 240acgttcggtc ggatcgactt tgcgcacaac aacgccggga ttgaaggcgt gcaggcaatg 300ctggccgatt atcccgaaga ggtctgggat cgggtgatcg agatcaacct caaaggggtc 360tggttgtgta tgaagtacga aatccggcac atgctcaagc agggtggcgg tgcgattgtg 420aatacctcat cggtcgccgg tctggccgga tcacgtggcg tttcggcgta tgtagccagc 480aagcacggta ttgttggtat taccaaagcg gcagcccttg agtatgcgcg taacggtatt 540cgtgtcaacg caatctgtcc aggtacgatt catactgcga tgatcgaccg ctttacccag 600ggtgatcccc aactgcttgc ccagttcgct gagggtgaac cgattggtcg gctcggctcg 660cctgaagagg tcgccaatgc ggtgatctgg ctctgctcag ataaggcttc gtttgtgacc 720ggagcgacac tggcggttga tggtggccgc ctggcgtaa 7597750DNARubrobacter xylanophilus DSM 9941 7atgctcgagg ggaaggtcgc ggtcatcacg ggggccggaa gcggcatagg ccgggccacc 60gcgctcaagt tcgcccgcga gggggcccgg gtcgtcgccg ccgagctcga cgagcgcggc 120ggggaggggg tggtccggga ggtgcgcagc ctcgggggcg aggcggtctt cgtccggacc 180gacgtctcgg agttcgcgca ggtggaggac gccgtcgagc gggcggtcgg ggagtacggc 240accctcgacg tgatgttcaa caacgccggc atcgggcact acgcccccct gctggagcac 300gagcccgagc actacgaccg ggtggtccgg gtgaaccagt acggcgtcta ctacgggata 360ctcgccgccg ggagaaagat ggtcgccctg aagaaccccg gcttgatcat caacaccgcc 420tcggtctacg ccttcctcgc ctcgccgggg gtcatcggct accacgccgc caagggggcg 480gtcaagatga tgacccaggc ggcggcgctg gagctcgccc cgcacggcat aagggtcgtc 540gccatcgccc cgggcggggt ggacaccccc atcatccagg gctacaagga catggggctc 600ggcgagaggc tggcccgcgg ccagatgcgc cgccggctcc agacccccga gcagatcgcc 660ggggcggtcg ccctgctcgc caccgacgag gccgacgcca taaacggctc ggtggtcatg 720accgacgacg gctacgcgga gttcaagtag 7508732DNACandida magnoliae CBS 6396 8atgtctgcta cttcgaacgc tcttatcact ggtgccagcc gcggaatggg cgaggccaca 60gctattaagc ttgcccttga ggggtacagc gtcacccttg catcacgcgg tattgagcag 120ctcaatgcca tcaaggaaaa actacccatc gtgaagaagg gccagcagca ctacgtttgg 180cagctcgatc ttagtgacat cgaggcggct tccaccttca agggggctcc tctgcctgcc 240agcagctacg acgtgttctt cagcaacgcc ggtgtggtgg actttgctcc gttcgcagac 300caaagcgaga ctgcgcaaaa ggacctgttc acggttaacc tgctgtcgcc tgttgcgttg 360accaagacca ttgttaaggc catcgccgac aagccccgcg agacgcctgc tcacattatc 420ttcacctcgt ccattgtcgg aattcgcggt gttcccaacg tggcggtcta cagcgccacc 480aagggcgcga ttgacagctt tgcgcgctcg cttgctcgtg agttcggtcc caagaacatc 540cacgttaact gcgtgaaccc gggcacgacg cgcaccgaga tgacaaaggg cgttgatctc 600gcggctttcg gcgatgttcc tatcaagggc tggatcgagg tcgatgcgat tgccgacgct 660gtgctgtttt tgatcaagtc caagaacatc actggccagt cgctcgttgt tgacaacgga 720ttcggtgttt aa 7329726DNACandida magnoliae DSM 70638 9atgacatcta cacctaatgc cctcatcacg ggaggcagcc gcggcattgg cgcttccgcc 60gccatcaaac tggctcaaga agggtacagc gtcacgctgg cgtcccgcga ccttgagaaa 120cttactgagg tcaaggacaa gctgccaatc gtgagaggtg gacagaaaca ctacgtttgg 180cagctcgatc ttgccgatgt ggaggctgca tcgtctttca aggcggctcc tctgccggcc 240agcagctacg atttgtttgt ttcgaacgcc ggaattgccc agttctcgcc tacggcagag 300catactaata gtgagtggct gaacattatg accattaact tagtgtcccc gattgccctg 360acgaaggctc ttttgcaggc cgtttctggg aggtcgagcg agaacccgtt tcagatcgtc 420ttcatctcgt cggttgcagc actacgtggc gttgcacaaa cggccgtcta cagtgcgtcg 480aaggctggta ctgatggatt cgcacgctca cttgctcgcg aactaggtcc tcaaggtgtt 540catgtgaacg tggtgaaccc tggctggact aagacagaca tgacggaagg agtcgaaacc 600ccaaaggaca tgcccattaa gggctggatc cagcctgagg caattgctga tgctgtagta 660ttccttgcga ggtcgaaaaa cattaccggc gcgaatattg tagtggacaa tggtttctcg 720acgtaa 72610726DNACandida magnoliae DSM 70638 10atgacgacta cttcaaacgc gcttgtcact ggaggcagcc gcggcattgg cgctgcctcc 60gccattaagc tggctcagga gggctacaat gttacgctgg cctctcgcag tgttgataaa 120ctgaatgaag taaaggcgaa actcccaatt gtacaggacg ggcagaagca ctacatttgg 180gaactcgatc tggctgatgt ggaagctgct tcgtcgttca agggtgctcc tttgcctgct 240cgcagctacg acgtctttgt ttcgaacgcg ggcgtcgctg cgttctcgcc cacagccgac 300cacgatgata aggagtggca gaacttgctt gccgtgaact tgtcgtcgcc cattgccctc 360acgaaggccc tcttgaagga tgtctccgaa aggcctgtgg acaagccact gcagattatc 420tacatttcgt cggtggccgg cttgcatggc gccgcgcagg tcgccgtgta cagtgcatct 480aaggccggtc ttgatggttt tatgcgctcc gtcgcccgtg aggtgggccc gaagggcatc 540catgtgaact ccatcaaccc cggatacacg aagactgaaa tgaccgcggg cattgaagcc 600cttcctgatt tgcctatcaa ggggtggatc gagcccgagg caattgctga cgcggttctg 660tttctggcaa agtccaagaa tatcaccggc acaaacattg tggtcgacaa tggcttgatt 720gcttaa 7261138DNAArtificial Sequencemisc_feature(1)..(38)primer 11ggaattccat atgatggagc cacctttcat tgggaagg 381234DNAArtificial Sequencemisc_feature(1)..(34)primer 12cccaagctta ttattacgcc aggcggccac catc 341334DNAArtificial Sequencemisc_feature(1)..(34)primer 13cccaagctta ttattacgcc aggcggccac catc 341435DNAArtificial Sequencemisc_feature(1)..(35)primer 14ggaattccat atgatgtctg ctacttcgaa cgctc 351533DNAArtificial Sequencemisc_feature(1)..(33)primer 15ccgctcgagt tattaaacac cgaatccgtt gtc 331635DNAArtificial Sequencemisc_feature(1)..(35)primer 16cacatgcatg cagatgtctg ctacttcgaa cgctc 351734DNAArtificial Sequencemisc_feature(1)..(34)primer 17gcccgagctc ttattaaaca ccgaatccgt tgtc 341812PRTArtificial Sequencemisc_feature(1)..(12)partial sequence ofoxidoreductase/dehydrogenase 18Asn Ala Leu Val Thr Gly Ala Ser Arg Gly Ile Gly1 5 101912PRTArtificial Sequencemisc_feature(1)..(12)partial sequence ofoxidoreductase/dehydrogenase 19Asn Ala Leu Val Thr Gly Gly Ser Arg Gly Ile Gly1 5 102012PRTArtificial Sequencemisc_feature(1)..(12)partial sequence ofoxidoreductase/dehydrogenase 20Asn Ala Leu Ile Thr Gly Gly Ser Arg Gly Ile Gly1 5 102112PRTArtificial Sequencemisc_feature(1)..(12)partial sequence ofoxidoreductase/dehydrogenase 21Asn Ala Leu Ile Thr Gly Ala Ser Arg Gly Ile Gly1 5 102212PRTArtificial Sequencemisc_feature(1)..(12)partial sequence ofoxidoreductase/dehydrogenase 22Asn Ala Leu Ile Thr Gly Gly Ser Arg Gly Met Gly1 5 102312PRTArtificial Sequencemisc_feature(1)..(12)partial sequence ofoxidoreductase/dehydrogenase 23His Ala Leu Val Thr Gly Ala Ser Arg Gly Ile Gly1 5 10247PRTArtificial Sequencemisc_feature(1)..(7)partial sequence ofoxidoreductase/dehydrogenase 24Gly Tyr Ser Val Thr Leu Ala1 5257PRTArtificial Sequencemisc_feature(1)..(7)partial sequence ofoxidoreductase/dehydrogenase 25Gly Tyr Asn Val Thr Leu Ala1 5267PRTArtificial Sequencemisc_feature(1)..(7)partial sequence ofoxidoreductase/dehydrogenase 26Gly Tyr Ser Val Thr Leu Val1 5277PRTArtificial Sequencemisc_feature(1)..(7)partial sequence ofoxidoreductase/dehydrogenase 27Gly Tyr Asn Val Thr Leu Val1 5288PRTArtificial Sequencemisc_feature(1)..(8)partial sequence ofoxidoreductase/dehydrogenase 28Phe Lys Gly Ala Pro Leu Pro Ala1 5298PRTArtificial Sequencemisc_feature(1)..(8)partial sequence ofoxidoreductase/dehydrogenase 29Phe Lys Ala Ala Pro Leu Pro Ala1 5306PRTArtificial Sequencemisc_feature(1)..(6)partial sequence ofoxidoreductase/dehydrogenase 30Phe Val Ser Asn Ala Gly1 5316PRTArtificial Sequencemisc_feature(1)..(6)partial sequence ofoxidoreductase/dehydrogenase 31Phe Phe Ser Asn Ala Gly1 5326PRTArtificial Sequencemisc_feature(1)..(6)partial sequence ofoxidoreductase/dehydrogenase 32Phe Val Cys Asn Ala Gly1 5336PRTArtificial Sequencemisc_feature(1)..(6)partial sequence ofoxidoreductase/dehydrogenase 33Phe Val Ala Asn Ala Gly1 5349PRTArtificial Sequencemisc_feature(1)..(9)partial sequence ofoxidoreductase/dehydrogenase 34Ser Pro Ile Ala Leu Thr Lys Ala Leu1 5359PRTArtificial Sequencemisc_feature(1)..(9)partial sequence ofoxidoreductase/dehydrogenase 35Ser Pro Val Ala Leu Thr Lys Thr Ile1 5369PRTArtificial Sequencemisc_feature(1)..(9)partial sequence

ofoxidoreductase/dehydrogenase 36Ser Pro Ile Ala Leu Thr Lys Thr Leu1 5379PRTArtificial Sequencemisc_feature(1)..(9)partial sequence ofoxidoreductase/dehydrogenase 37Ser Pro Val Ala Met Thr Lys Ala Leu1 5389PRTArtificial Sequencemisc_feature(1)..(9)partial sequence ofoxidoreductase/dehydrogenase 38Ser Gln Ile Ala Leu Thr Lys Ala Leu1 5397PRTArtificial Sequencemisc_feature(1)..(7)partial sequence ofoxidoreductase/dehydrogenase 39Ala Val Tyr Ser Ala Ser Lys1 5407PRTArtificial Sequencemisc_feature(1)..(7)partial sequence ofoxidoreductase/dehydrogenase 40Ala Val Tyr Ser Ala Thr Lys1 5416PRTArtificial Sequencemisc_feature(1)..(6)partial sequence ofoxidoreductase/dehydrogenase 41Pro Ile Lys Gly Trp Ile1 5426PRTArtificial Sequencemisc_feature(1)..(6)partial sequence ofoxidoreductase/dehydrogenase 42Pro Ile Ser Gly Trp Ile1 5

Claims

1. A process for the enantioselective enzymatic reduction of secodione derivatives of general formula I ##STR00008## wherein the ring structures comprise no, one or several heteroatoms,R₁ is hydrogen or a C₁-C₄ alkyl group,R₂ is hydrogen, a C₁-C₈ alkyl group or a protective group for OH known in prior art, such as an ester,R₃ is hydrogen, a methyl group or a halide,the structural element ##STR00009## represents a benzene ring or a C₆ ring having 0, 1 or 2 C--C double bonds,a double bond is optionally included at positions 6/7 or 7/8, andthe carbon at positions 1, 2, 4, 5, 6, 7, 8, 9, 11, 12 and 16 is independently substituted with hydrogen, a C₁-C₄ alkyl group, a halide or a phenyl group,wherein the secodione derivative is reduced with an oxidoreductase/dehydrogenase in the presence of NADH or NADPH as a cofactor,characterized in that the secodione derivative is used in the reaction batch at a concentration of ≧10 g/l and the oxidized cofactor NAD or NADP formed by the oxidoreductase/dehydrogenase is regenerated continuously.

2. A process for the enantioselective enzymatic reduction of secodione derivatives of general formula I, wherein the secodione derivative is reduced with an oxidoreductase/dehydrogenase in the presence of NADH or NADPH as a cofactor, characterized in that the oxidoreductase/dehydrogenasea) comprises an amino acid sequence in which at least 50% of the amino acids are identical to those of amino acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5,b) is encoded by the nucleic acid sequence SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, orc) is encoded by a nucleic acid sequence which hybridizes to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 under stringent conditions.

3. A process for the enantioselective enzymatic reduction of secodione derivatives of general formula I, wherein the secodione derivative is reduced with an oxidoreductase/dehydrogenase in the presence of NADH or NADPH as a cofactor, characterized in that the oxidoreductase/dehydrogenase has a length of from 230 to 260 amino acids and comprises one or several of the partial sequences selected from the group consisting of [sequences SEQ ID NO:18 to SEQ ID NO:42]nalvtgasrgig, nalvtggsrgig, nalitggsrgig, nalitgasrgig, nalitggsrgmg, halvtgasrgig,gysvtla, gynvtla, gysvtlv, gynvtlv,fkgaplpa, fkaaplpa,fvsnag, ffsnag, fvcnag, fvanag,spialtkal, spvaltkti, spialtktl, spvamtkal, sqialtkal,avysask, avysatk,pikgwi and pisgwi.

4. A process according to claim 1, characterized in that the oxidoreductase/dehydrogenasea) comprises an amino acid sequence in which at least 50% of the amino acids are identical to those of amino acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5,b) is encoded by the nucleic acid sequence SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, orc) is encoded by a nucleic acid sequence which hybridizes to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 under stringent conditions.

5. A process according to claim 1, characterized in that the oxidoreductase/dehydrogenase has a length of from 230 to 260 amino acids and comprises one or several of the partial sequences selected from the group consisting of [sequences SEQ ID NO:18 to SEQ ID NO:42]nalvtgasrgig, nalvtggsrgig, nalitggsrgig, nalitgasrgig, nalitggsrgmg, halvtgasrgig,gysvtla, gynvtla, gysvtlv, gynvtlv,fkgaplpa, fkaaplpa,fvsnag, ffsnag, fvcnag, fvanag,spialtkal, spvaltkti, spialtktl, spvamtkal, sqialtkal,avysask, avysatk,pikgwi and pisgwi.

6. A process according to claim 2 or 3, characterized in that the oxidized cofactor NAD or NADP formed by the oxidoreductase/dehydrogenase is regenerated continuously.

7. A process according to any of claims 1 to 3, characterized in that the oxidized cofactor NAD or NADP is regenerated by oxidation of an alcohol.

8. A process according to claim 7, characterized in that a secondary alcohol having the general formula R_XRYCHOH is used for cofactor regeneration, wherein R_X and R_Y independently of each other are hydrogen, a branched or unbranched C₁-C₈ alkyl group and C_total≧3.

9. A process according to claim 7, characterized in that an alcohol from the group consisting of 2-propanol, 2-butanol, 2-pentanol, 4-methyl-2-pentanol, 2-hexanol, 2-heptanol, 5-methyl-2-hexanol, cyclohexanol or 2-octanol is used for cofactor regeneration.

10. A process according to any of claims 1 to 3, characterized in that an oxidoreductase/dehydrogenase is additionally added for the regeneration of the cofactor.

11. A process according to any of claims 1 to 3, characterized in that the TTN (total turn over number=mol of reduced secodione derivative/mol of cofactor used) is ≧10.sup.3.

12. A process according to any of claims 1 to 3, characterized in that it is carried out in an aqueous organic two-phase system.

13. A process according to any of claims 1 to 3, characterized in that an organic solvent, preferably diethyl ether, tertiary butyl methyl ether, diisopropyl ether, dibutyl ether, ethyl acetate, butyl acetate, heptane, hexane, toluene, dichloromethane, cyclohexane or mixtures thereof, is additionally used.

14. A process according to any of claims 1 to 3, characterized in that the secodione derivative is used in the reaction batch in an amount of from 10 g/l to 500 g/l, based on the total volume, preferably from 25 g/l to 300 g/l, particularly preferably from 50 g/l to 200 g/l.

15. A process according to any of claims 1 to 3, characterized in that 13-ethyl-3-methoxy-8,14-seco-gona-1,3,5(10),9(11)-tetraene-14,17-dione (ethyl secodione--Formula III) is used as the secodione derivative.

16. A process according to any of claims 1 to 3, characterized in that 13-methyl-3-methoxy-8,14-seco-gona-1,3,5(10),9(11)-tetraene-14,17-dione (methyl secodione--Formula II) is used as the secodione derivative.

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