Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P33/00—Preparation of steroids
  • C12P33/06—Hydroxylating
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • 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)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
  • C12Y114/14—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen (1.14.14)

Definitions

• the present invention relates to agents and methods for the hydroxylation of steroids.
• 3,7,12-trihydroxylated bile acids such as, for example, cholic acid (3 ⁇ , 7 ⁇ , 12 ⁇ -trihydroxy-5 ⁇ -cholanic acid) or ursocholic acid (3 ⁇ , 7 ⁇ , 12 ⁇ -trihydroxy-5 ⁇ -cholanic acid) are industrially important Chemicals, including as raw materials for the production of ursodeoxycholic acid (UDCA).
• Ursodeoxycholic acid is used as a drug, among other things, to dissolve smaller X-ray negative gallstones and to treat liver diseases, primarily ciliary cirrhosis and primarily sclerosing cholangitis.
• bile is an aqueous mixture of bile acids, lipids, cholesterol and other substances, the separation of the constituents in the production of bile acids is of particular importance.
• the bile acids also represent a mixture, the components of which differ in the number and position of the hydroxyl groups.
• bovine bile also contains a significant proportion of deoxycholic acid, which differs from cholic acid in the absence of the OH group at position 7 (3a, 12 ⁇ -dihydroxy-5 ⁇ -cholanic acid).
• Deoxycholic acid has a much lower level commercial value as 3,7,12-trihydroxylated bile acids. There is therefore an industrial interest in converting deoxycholic acid into a 3,7,12-trihydroxylated bile acid by the targeted introduction of a hydroxyl group at position 7.
• Schmitz et al. (Microbial Cell Factories 13 (1): 1-13 (2014)) describe the production of 7-hydroxyl derivatives of dehydroepiandrosterone (DHEA) or pregnenolone (PREG) by reaction with a cytochrome P450 monooxygenase.
• DHEA dehydroepiandrosterone
• PREG pregnenolone
• CN 102002 518 B discloses the conversion of 3-ß-cholesterol acetate to 7-ß-hydroxyl-3-ß-cholesterol acetate with a hydroxylase.
• WO 2020/109776 A2 discloses a method for the hydroxylation or dealkylation of various organic compounds, including the steroid pregenenolone, with a cytochrome P450.
• Xi and X2 independently of one another H, CI, F, Br, I, CF 3 , a Ci to C 6 alkyl radical, OH, a Ci to C 6 alkoxy radical, CN, N0 2 , N (R 6) 2, an epoxy group, CHO or a C0 2 R. 6 radical, where
• Ri and R 2 are independently H, OH, OR7 or 0, where
• R 3 H, OH, OR 8 a Ci to C10 alkyl radical, a Ci to C10 alkenyl radical, -CHO, -C (O) (CH 3) , -C (O) (CH 2 OH), - CH (CH 3 ) C (O) CH 3, -CH (CH 3) ((CH 2) 2CO2R9) or - CH, is (CH 3) ((CH 2) 2CONHR9) wherein
• R4 is H, OH, or -OR10, where
• R 5 is H, CF 3 , a Ci to C 6 alkyl radical, a Ci to C 6 alkenyl radical, OH, 0, or a Ci to C 6 alkoxy radical, the dashed line denoting an optional double bond, with the proviso that the B-ring has no double bond when the A-ring has a C4-C5 double bond and the C-ring has no double bond when Xi and X2 form an epoxy group, comprising the step of reacting a 7-deoxystero- ids with the general formula (II) with a cytochrome P450 hydroxylase or a functional variant thereof in the presence of at least one redox partner system and a system for regenerating the redox partner system, the cytochrome P450 enzyme comprising an amino acid sequence that is at least 90%, in particular 100%, identical is solved with the amino acid sequence SEQ ID No.
• cytochrome P450 and functional fragments thereof are able to hydroxylate steroids such as cholic acid or derivatives thereof with the formula (I) at position 7.
• a system for regenerating the redox partner system By coupling this reaction with at least one redox partner system and a system for regenerating the redox partner system, the equilibrium of the reaction can be shifted to the side of the end product and thus its yield can be increased significantly.
• the regeneration of the redox partner system can take place in the presence of a second system (a regeneration system) which preferably comprises at least one oxidoreductase and at least one substrate of the at least one oxidoreductase.
• Cytochrome P450 and functional variants thereof which require the oxidation of reduction equivalents NAD (P) H are surprisingly able to selectively attack 7-deoxysteroids, such as 7-deoxycholic acid, and derivatives thereof To hydroxylate position 7.
• the cytochrome P450 enzyme comprises an amino acid sequence which is at least 90%, in particular 100%, identical to the amino acid sequence SEQ ID No. 1 or 2.
• Cytochromes P450 catalyze monooxygenase reactions of a large number of endogenous and exogenous substrates. They are involved in the metabolism of steroids, eicosanoids, fatty acids and bile acids as well as exogenous substrates such as drugs, insecticides and chemical carcinogens.
• Cytochromes P450 according to the present invention can be used, for example, from bacteria, such as, for example, actinobacteria, in particular, for example, of the genus Streptomyces.
• the sequences can be isolated with the aid of known techniques, for example from genomic DNA or a cDNA library.
• the cytochromes P450 according to the present invention or their functional variants can optionally be present in their original organism or isolated from it, or they are expressed recombinantly or synthetically. According to the invention, recombinantly expressed polypeptides are preferably used.
• E. coli Escherichia coli
• Bacillus subtilis Bacillus subtilis
• Saccharomyces cerevisiae Saccharomyces cerevisiae
• Pichia pastoris can be used for the recombinant expression of enzymes according to the present invention.
• Corresponding protocols are described in detail in the relevant specialist literature or are known to a competent person.
• enzymes / polypeptides are preferably used as proteins recombinantly overexpressed in E. coli, the corresponding cell lysates preferably either without further processing / purification or after relatively simple processing steps (eg centrifugation, precipitation, concentration or freeze drying).
• E. coli cells can also be used in the reaction after recombinant overexpression of the enzymes used without cell disruption or, for example, after a freezing / thawing cycle.
• Suitable expression plasmids are known to a person skilled in the art and many of them can be obtained commercially.
• “Functional variants” of cytochrome P450 can be fragments or mutation variants of cytochrome P450, fragments of cytochrome P450 also being referred to as "functional fragments". "Functional variants" of cytochrome P450 are able to catalyze the same reaction as the protein from which they were derived. Whether a variant is functional, ie whether it catalyzes the same reaction as the protein from which it is derived can be determined by establishing that the variant catalyzes the same reaction. lated methods in the prior art or those described therein. The conversion rates of substrates by the functional variants according to the invention can differ from the conversion rates of the cytochrome P450 from which they were derived.
• Derivatives of 7-deoxysteroids include compounds derived from 7-deoxysteroids with a wide variety of modifications as defined above.
• Xi, X2, R 4 and R 5 are H and Ri and R2, independently of one another, are H, OH, OR 8 or 0, where R 8 is -C (O) H, -C (O) CH 3 , -C (O) CH 2 CH 3 , -C (O) (CH 2) CH 3 , -C (O) CH (CH 3) 2, -C (O) (CH 2) 3 CH 3 , -C (O) CH (CH 3) CH 2 CH 3 , - C (O) CH2CH2 (CH 3) 2, -C (O) C (CH 3) 3 , -C (O) Ph, - C (O) CH 2 Ph is
• R 3 is a Ci to C 10 alkyl radical, a Ci to C 10 alkylene radical, - CH (CH 3 ) ((CH 2 ) 2CO2R9) or -CH (CH 3 ) ((CH 2 ) 2CONHR9), where R g -CH 3 , -CH 2 COOH, -CH 2 CH 3 , -CH (CH 3 ) 2 , - (CH 2 ) 2 CH 3 , - (CH 2 ) 2 SO 3 H, C (CH 3 ) 3 , - (CH 2 ) 3 is CH 3 , -CH (CH 3 ) CH 2 CH 3 , -CH2CH2 (CH 3 ) 2, an aryl group or an alkylary group.
• the aryl group is selected from the group consisting of a phenyl radical, one with F,
• the alkylaryl group is selected from the group consisting of a benzyl group, a halogenated benzyl group, the halogen being F, Ci or Br, and a benzyl group substituted by NO 2.
• Ri is OH, R 2 O or OH
• R 3 is CH (CH 3) (( CH 2) 2 CO2R5)
• R4 is H
• R 5 is H.
• the 7-deoxysteroid is with the general formula (II) selected from the group consisting of 3a, 12 ⁇ -dihydroxy-5ß-cholan-24-acid, 3a, 12ß-dihydroxy-5ß-cholan-24-acid, 3ß, 12 ⁇ -dihydroxy-5ß-cholan - 24-acid, 3ß, 12ß-dihydroxy-5ß-cholan-24-acid, 3ß-hydroxy-12-keto-5ß-cholan-24-acid, 3-keto, 12ß-hydroxy-5ß-cholan-24 Acid, 3-keto, 12 ⁇ -hydroxy-5ß-cholan-24-acid, 3 ⁇ -hydroxy-5ß-cholan-24-acid, 3-keto-5ß-cholan-24-acid, 3ß-hydroxy-5ß-cholan -24 acid and ester of the respective acid.
• the general formula (II) selected from the group consisting of 3a, 12 ⁇ -dihydroxy-5ß-cholan-24-acid, 3a, 12ß-dihydroxy-5ß-cholan-24-acid, 3
• the cytochrome P450 enzyme which is used according to the invention for the hydroxylation of 7-deoxysteroids and derivatives thereof with the general formula (II) to give a steroid or a derivative thereof with the general formula (I) comprises a Amino acid sequence which is at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%, in particular 100%, is identical to the amino acid sequence SEQ ID No. 1 or 2.
• SEQ ID No. 2 Amino acid sequences SEQ ID No. 1 and 2 are preferably encoded by nucleic acid sequences SEQ ID No. 3 and 4, nucleic acid sequences SEQ ID No. 5 and 6 being optimized for expression in E. coli.
• Ident means that two or more amino acid sequences, if these are superimposed on one another, can have a certain “identity” (matching amino acid residues at identical positions) to one another. "Identity” is defined in this invention as the percentage of the amino acids of the amino acid sequences in question that are identical to the amino acids of the starting sequence, after aligning the two sequences and the introduction of gaps, if necessary, by the to achieve maximum sequence identity in percent, as demonstrated by the "Protein BLAST" program (blastp; Altschul et al., J. Mol. Biol.
• blastp Protein-Protein-BLAST
• word size 6
• Matrix BLOSUM62
• Gap costs "Existence” 11, “Extension”1; conditional compositional score matrix adjustment; no filter and none Mask.
• a percentage (%) value for the amino acid sequence identity is determined by the number of matching identical nucleotides divided by the sequence length for which the percentage identity is recorded.
• the Cy - tochrom P450 enzyme comprises an amino acid sequence which is at least 90%, in particular 100%, identical to the amino acid sequence SEQ ID No. 1 or 2. This conversion takes place in the presence of redox partners or a redox partner system which is in is able to provide electrons for the hydroxylation reaction.
• the at least one redox partner system comprises
• the redox partner system used according to the invention can ferredoxin, ferredoxin reductase and NAD (P) H; Cytochrome P450 reductase and NAD (P) H; or NAD (P) H alone, a redox partner system comprising ferredoxin, ferredoxin reductase and NAD (P) H being particularly preferred.
• NAD + denotes the oxidized form and NADH the reduced form of nicotinamide adenine dinucleotide
• NADP + denotes the oxidized form and NADPH the reduced form of nicotinamide adenine dinucleotide phosphate
• concentration of the redox cofactors NAD (P) + and / or NAD (P) H in a reaction mixture is preferably between 0.001 mM and 10 mM, even more preferably between 0.05 mM and 1 mM.
• Ferredoxins which can be regenerated in the presence of NAD (P) + and at least one ferredoxin reductase are particularly preferably used as redox partners.
• the at least one ferredoxin is selected from the group consisting of adrenodoxins, putidaredoxins and flavodoxins, combinations thereof also optionally being used.
• a possible pair of redox partners preferably comprises putidaredoxin and putidaredoxin reductase from Pseudomonas putida.
• a skilled person is also able to identify further ferredoxin proteins and ferredoxin reductases which are potential redox partners for the cytochrome P450 according to the invention.
• the suitability as a redox partner can be checked in a functional assay, as described in Examples 3 to 5, for example.
• the putidaredoxin used in these examples and / or the putidaredoxin reductase used therein can be replaced by possible alternative proteins or enzymes. If sufficient formation of the desired product (e.g. ursocholic acid) is observed, the tested redox partners can be viewed as functional alternatives to putidaredoxin and / or putidaredoxin reductase.
• the ferredoxin used in the method according to the invention comprises an amino acid sequence which is at least 80%, preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%, in particular 100%, is identical to the amino acid sequence SEQ ID No. 7, where X is a methionine residue or not an amino acid.
• the at least one ferredoxin reductase is selected from the group of flavodoxin reductases and putidaredoxin reductase.
• the ferredoxin oxidized in the course of the hydroxylation reaction according to the invention can be reduced with the aid of a ferredoxin reductase and NAD (P) H.
• reduced ferredoxin is made available again or regenerated for a further hydroxylation reaction of the substrate according to the invention with consumption of NAD (P) H.
• the ferredxone reductase can be a flavodoxin reductase and / or a putidaredoxin reductase.
• the ferredoxin reductase used in the method according to the invention comprises an amino acid sequence which is at least 80%, preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% , even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%, in particular 100%, is identical to the amino acid sequence SEQ ID No. 8.
• Amino acid sequences SEQ ID No. 7 and 8 are preferably replaced by nucleic acid sequences SEQ ID No. 9 or
• nucleic acid sequences SEQ ID No. 11 and 12 are optimized for expression in E. coli.
• cytochrome P450 any ferredoxins and ferredoxin reductases in bacteria, in particular in E. coli, is particularly advantageous when using nucleic acids with the nucleic acid sequences SEQ ID No. 5, SEQ ID No. 6 , SEQ ID No. 11 and / or SEQ ID No. 12. Therefore, further aspects of the present invention relate to a nucleic acid (DNA and / or RNA) with a nucleic acid sequence selected from the group consisting of SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 11 and SEQ ID No. 12 and vectors and / or cells, in particular E. coli cells, comprising at least one of these sequences.
• ferredoxins and ferredoxin reductases are expressed (co-expressed) together with cytochrome P450 in a production strain (e.g. E. coli strain).
• the ferredoxins, ferredoxin reductases and the cytochrome P450 can also be expressed separately from one another. It is also advantageous to express ferredoxin and cytochrome P450 or ferredoxin reductase and cytochrome P450 together.
• the at least one oxidoreductase is selected from the group consisting of oxidoreductase (EC: 1.1.1), aldehyde dehydrogenase (EC: 1.2.1), amino acid dehydrogenase (EC: 1.4 .1), flavin reductase (EC: 1.5.1), transhydrogenase (EC: 1.6.1), nitrite reductase (EC: 1.7.1) and phosphonate dehydrogenase (EC: 1.20.1) is, preferably selected from the group consisting of alcohol dehydrogenase, hydroxysteroid dehydrogenase, phosphite dehydrogenase and sugar dehydrogenase
• the cofactor used NADH / NAD + and / or NADPH / NADP +
• a system for regenerating the redox partner system advantageously oxidoreductases, to the reaction mixture.
• Oxido-reductases convert substrates through reduction and oxidation, whereby in the course of these reactions NADH is oxidized to NAD + or NADPH to NADP + or NAD + is reduced to NADH or NADP + to NADPH.
• the system for regenerating the redox partner system therefore preferably comprises at least one oxidoreductase and at least one substrate of the at least one oxidoreductase.
• the oxido reductase used in the process according to the invention is preferably an alcohol and / or sugar dehydrogenase.
• the oxidoreductase is selected from the group consisting of glucose dehydrogenase, glucose-6-phosphate dehydrogenase, arabinose dehydrogenase, xylose dehydrogenase, sorbitol dehydrogenase, xylitol dehydrogenase hydrogenase, 12 ⁇ -hydroxysteroid dehydrogenase, 7 ⁇ -hydroxysteroid dehydrogenase, 20 -hydroxysteroid dehydrogenase, 17 ⁇ -hydroxysteroid dehydrogenase, 17 -hydroxysteroid dehydrogenase, 3 ⁇ -hydroxysteroid dehydrogenase, 3 ⁇ -hydroxysteroid dehydrogenase, 1l ⁇ -hydroxysteroid dehydrogenase, 1l ⁇ -hydroxysteroid dehydrogenase Dehydrogenase and formate dehydrogenase.
• the reaction mixture can comprise at least one oxidoreductase and one hydroxylase.
• the reaction mixture therefore comprises at least one substrate of the at least one oxidoreductase selected from the group consisting of arabinose, xylose, glucose, sorbitol, xylitol, cholan-24 acid,
• the process according to the invention is even more preferred at a temperature from 10 ° C to 40 ° C, preferably from 15 ° C to 38 ° C, even more preferably from 20 ° C to 30 ° C from 22 ° C to 26 ° C.
• the enzyme activity of cytochrome P450 for the reaction according to the invention is particularly high in this range.
• the method according to the invention is carried out at a pH of 6.5 to 8.5, preferably from 7 to 8, even more preferably from 7.2 to 7.8.
• the enzyme activity of cytochrome P450 is highest at this pH value in order to ensure a corresponding conversion of the substrate.
• hydroxylation of deoxysteroids or deoxysteroid derivatives can take place regioselectively at position 7 of the basic steroid structure.
• a 7bet ⁇ -hydroxyl group can be introduced stereoselectively, so that, for example, ursocholic acid and / or ursocholic acid derivatives can be produced.
• the process according to the invention is carried out in the presence of at least one organic solvent.
• a single organic solvent is preferably used, so that a single-phase system is present. It is also possible to use a mixture of two or more organic solvents which, according to the invention, are miscible with one another, so that a single-phase system is present.
• the organic solvent can be protic or aprotic, with ⁇ -protic solvents being preferred.
• organic solvents examples include alcohols, ethers, esters, glycols, ketones, amides, sulphoxides, organic acids, cycloalkanes, aromatics and chlorinated hydrocarbons.
• suitable organic solvents are methanol, ethanol, isopropanol, 2-butanol, 4-methyl-2-pentanol (methyl Isobutyl alcohol, MIBA), diethyl ether (Et 2 0), diisopropyl ether (iPr 2 O), dioxane, tetrahydrofuran (THF), 2-methyl-tetrahydrofuran (Me-THF), ethyl acetate, ethylene glycol, methyl isobutyl ketone (MIBK ), 2-butanone, acetone, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), cyclohexane, toluene, trich
• Suitable mixtures are, for example, mixtures of hexane and ethyl acetate or isopropanol, as well as mixtures of trichloromethane and phenol.
• the present invention is not restricted to the above exemplary list of solvents.
• the organic solvent is preferably an aprotic organic solvent, particularly preferably a solvent selected from the group consisting of dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and dimethyl acetamide (DMA).
• DMF dimethylformamide
• DMSO dimethyl sulfoxide
• DMA dimethyl acetamide
• the amount of the organic solvent is chosen such that the compound of the general formula (II) is completely dissolved and the enzyme activity is retained.
• the compound of the formula (II), for example lithocholic acid, is preferably dissolved in the organic solvent up to the solubility limit.
• the substrate of the enzyme is placed in the organic solvent.
• the isolation of the product can be carried out in different ways.
• the product can be extracted from the reaction mixture using a suitable organic solvent. Depending on the substrate, such solvents are described in the literature.
• Cholic acids and their derivatives according to the present invention can be isolated from reaction mixtures, for example with ethyl acetate, if necessary after acidifying the reaction mixture, for example with HCl.
• a special case is a process in which bile acids are present in the form of a salt, for example a sodium salt in aqueous solution.
• the product can be precipitated by acidifying the reaction mixture.
• a sufficient amount of, for example, HCl or dilute HCl can be added to the reaction mixture.
• the product is predominantly in the form of a suspension.
• the product can then be removed from the reaction mixture by common methods such as filtration or centrifugation.
• Another alternative that can be used, for example, for product isolation are chromatographic methods such as affinity chromatography or ion exchange chromatography. It is also possible, for example, to obtain product by evaporating the reaction solvent.
• the product (s) can also remain in the reaction mixture after the reaction, e.g. in order to carry out further reactions and, if necessary, to isolate an end product after these reactions have been completed. It is also conceivable that the substrate (s) for the method according to the present invention are generated by previous or parallel reactions in the same reaction batch.
• nucleic acid construct comprising a nucleic acid molecule coding for a cytochrome P450 enzyme as defined above at its 3 'end and / or 5' end directly or via a spacer at least one nucleic acid molecule coding for a polypeptide selected from the group consisting of a ferredoxin, a ferredoxin reductase and an oxidoreductase is bound.
• the nucleic acid constructs according to the invention are particularly suitable for the production of a steroid as defined at the outset.
• the cytochrome P450 enzyme and at least one protein selected from the group consisting of ferredoxins and ferredoxin reductases and oxidoreductases starting from a nucleic acid construct, it is possible to produce these enzymes or proteins in an amount that is necessary for an efficient implementation of the method according to the invention. It is particularly advantageous if all of these proteins are expressed on the nucleic acid construct according to the invention under the control of the same promoter.
• Another aspect of the present invention vector comprising a nucleic acid construct according to the present invention.
• the nucleic acid molecule coding for a cytochrome P450 enzyme can either be bound directly or via a spacer or a spacer sequence to further nucleic acid molecules which code for enzymes or proteins which can be used in the method according to the invention.
• the advantage of such a construct is that such a construct makes it possible to express the enzymes and proteins used in the method according to the invention, in particular cytochrome P450 and ferredoxin and / or ferredoxin reductase, in a comparable amount .
• Spacer or "spacer sequence”, as used here, is a nucleic acid sequence which has neither a stop codon nor any other functional motif.
• a spacer or a spacer sequence serves as a spacer between two ORFs in order, if necessary, to improve the transcription of these ORFs.
• the cytochrome P450 enzyme is encoded by a nucleic acid which is at least 90%, in particular 100%, identical to the nucleic acid sequence SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6.
• the ferredoxin comprises the amino acid sequence SEQ ID No. 7.
• the ferredoxin reductase is one Adrenodoxin reductase, preferably a putidaredoxin reductase.
• the ferredoxin reductase preferably comprises the amino acid sequence SEQ ID No. 8.
• Another aspect of the present invention relates to a vector comprising a nucleic acid construct according to the present invention.
• the vector according to the invention can be a cloning or expression vector and, depending on the organism in which it is introduced, can have corresponding sections in order, for example, to enable the transcription of an ORF.
• Another aspect of the present invention relates to a host cell comprising a nucleic acid construct according to the present invention.
• the host cell according to the invention can be used for cloning or for expressing the recombinantly introduced ORFs located on a nucleic acid construct.
• the lysate of such a host cell provided that it has expressed at least one of the enzymes or proteins required in the method according to the invention, intracellularly or extracellularly, can be used in the method according to the invention.
• the 7-deoxy steroid or a derivative thereof with the general formula (II) is preferably brought into contact with a cell suspension or cells in a culture supernatant and / or a lysate of a host cell according to the present invention.
• Example 1 Test of bacterial strains
• the following bacterial strains were obtained from the German strain collection for microorganisms and cell cultures (DSMZ): Saccharothrix longispora (DSM-43749), Catellatospora citrae (DSM-44097), Streptomyces hygroscopicus subsp. hygroscopicus (DSM-40578) and Asanoa ferruginea (DSM-44099).
• the strains were cultivated under standard conditions as recommended by DSMZ. As soon as the growth of the cultures had led to a visible cloudiness, deoxycholic acid (0.5 mM) was added and cultivation continued for up to 72 h.
• Example 3 Cloning Expression System Using the restriction enzyme Xhol, the following construct comprising coding regions for putidaredoxin reductase (PtR) and putidaredoxin (Ptx) was cloned into the plasmid pJ411 (DNA 2.0).
• PtR putidaredoxin reductase
• Ptx putidaredoxin
• Synthetic DNA (Life Technologies): 5 ', Xhol interface, HindIII interface, approx. 50 bp spacer DNA, ribosome binding site (rbs), ORF (open reading frame) putidaredoxin reductase (PtR ), approx. 50 bp spacer DNA, rbs, ORF putidaredoxin (Ptx), Xhol site, 3 '.
• the result of the cloning step was checked by means of restriction enzyme digestion and DNA sequencing.
• the expression plasmids produced with the identified P450 candidates can be used for the joint expression of the respective P450 proteins together with putidaredoxin reductase and putidaredoxin.
• the 3 ORFs of the respective expression plasmids are expressed under the control of a T7 promoter on a common mRNA but as separate polypeptides.
• the constructs of the P450 candidates to be tested were transformed into the E. coli expression strain BL21 (DE3). Overnight cultures were inoculated from individual colonies (LB (lysogeny broth) + kanamycin). The next day, 1: 100 expression cultures were inoculated with it (150 ml of TB (terrific broth) -P450 expression medium) and shaken in Schiskan flasks (1 L) initially at 37 ° C. for 3 h. The temperature was then lowered to 24 ° C. and shaking continued for 22 h. The cultures were harvested by centrifugation at 5000 g for 10 min, washed once with 0.9% (w / v) NaCl and pellets frozen at -80 ° C.
• the reactions were set up in 1.5 ml screw-top bottles and sealed with a lid with aluminum foil. The film was pierced in several places. It was shaken gently for 18 h at 24 ° C. 200 ml of the reaction mixture were diluted with 600 ml acetonitrile / 5 ml H 3 PO 4 (50%) and incubated at 55 ° C. for 15 minutes. The samples were then centrifuged for 5 minutes at 20817 rcf and analyzed by means of HPLC / DAD (eg Agilent 1200 Series;
• a redox cofactor (NADH) is oxidized by the P450 / Ptx / PtR reaction.
• NADH a redox cofactor
• a redox cofactor (NADH) is oxidized by the P450 / Ptx / PtR reaction.
• the cofactor regeneration here for example by means of the sugar dehydrogenase arabinose dehydrogenase
• the re- doxcofactor is reduced to its original state (whereby arabinose is oxidized to arabinolactone / arabonic acid). This enables the use of a substoichiometric amount of redox cofactor.
• Example 7 Examples: conversion depends on the cofactor concentration conversion with cofactor recycling
• the reactions were set up in 1.5 ml screw-top bottles and sealed with a lid with aluminum foil. The film was pierced in several places. It was shaken gently for 18 h at 24 ° C.
• NADH was recovered by sorbitol or xylitol dehydrogenase in the presence of sorbitol and NAD + .
• the deoxycholic acid used was quantitatively (100% conversion) to original socholic acid implemented.
• the identity of the product ursocholic acid was verified by GC / MS analysis and by 2D NMR.
• NADH redox cofactor
• the reactions were set up in 1.5 ml screw-top bottles and closed with a lid with aluminum foil. The film was pierced in several places. It was shaken gently for 18 h at 24 ° C. NADH was recovered by sorbitol or xylitol dehydrogenase in the presence of sorbitol and NAD + .
• solubility limits of LCA and UDCA in various organic solvents were determined. For this purpose, 10 mg or 100 mg LCA or UDCA were placed in a 15 mL flask. The organic solvent was added in steps of 100 ⁇ L and the mixture was treated by shaking in a vortex shaker and, if necessary, in an ultrasonic bath. It was assessed visually whether a clear solution was present.
• Example 10 Conversion of lithocholic acid to ursodeoxycholic acid with increased substrate concentration in the presence of an aprotic solvent
• NADH was recovered by sorbitol or xylitol dehydrogenase in the presence of sorbitol and NAD + .
• the reaction mixture was completely evaporated in a stream of air and redissolved with 1.1 ml of IPA + 0.5% TFA.
• the samples were then centrifuged for 5 minutes at 20817 rcf and the supernatant was analyzed by HPLC / RID (eg Agilent 1200 Series; column: Phenomenex Luna® Silica 100 ⁇ , 250 x 4.6 mm, 5 gm; flow rate: 1.0 ml / min, n-hexane / IPA 4: 1 + 0.05% TFA isocratic).
• HPLC / RID eg Agilent 1200 Series; column: Phenomenex Luna® Silica 100 ⁇ , 250 x 4.6 mm, 5 gm; flow rate: 1.0 ml / min, n-hexane / IPA 4: 1 + 0.05% TFA isocratic.

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