Methods for hydroxylating steroids

Abstract

The present invention relates to a method for preparing steroids, which comprises converting a 7-deoxysteroid with a cytochrome P450 enzyme or a functional variant thereof in the presence of at least one redox partner system and a system for regenerating the redox partner system.

Description

[Technical Field]

[0001] The present invention relates to means and methods for hydroxylation of steroids. [Background technology]

[0002] 3,7,12-Trihydroxylated bile acids, such as cholic acid (3α,7α,12α-trihydroxy-5β-cholanic acid) or ursocholic acid (3α,7β,12α-trihydroxy-5β-cholanic acid), are industrially important chemicals, particularly as starting materials for the production of ursodeoxycholic acid (UDCA). Ursodeoxycholic acid is used, in particular, to dissolve minor radiographically negative gallstones and as a pharmaceutical for the treatment of primary ciliary cirrhosis and primary sclerosing cholangitis in liver diseases.

[0003] The most important industrial source of 3,7,12-trihydroxylated bile acids is bile from gallbladders, which accumulate as slaughterhouse waste in meat production. Bovine bile is frequently used, in addition to bile from other animal species. There is no industrially relevant total synthesis of 3,7,12-trihydroxylated bile acids. Because bile acid production is linked to another product (meat), the response to increasing demand may be very limited. Therefore, utilizing raw bile as efficiently as possible is of great interest.

[0004] Since bile is an aqueous mixture of bile acids, lipids, cholesterol, and other substances, the separation of components during the extraction of bile acids is particularly important. Bile acids also constitute a mixture in which their components differ in the number and position of hydroxyl groups. In addition to cholic acid, bovine bile also contains a significant proportion of deoxycholic acid, which differs from cholic acid in that it lacks an OH group at position 7 (3α,12α-dihydroxy-5β-cholanic acid). Deoxycholic acid has far less commercial value than 3,7,12-trihydroxylated bile acid. Therefore, there is industrial interest in converting deoxycholic acid to 3,7,12-trihydroxylated bile acid by selectively introducing a hydroxyl group at position 7.

[0005] During hydroxylation, the oxygen atom is formally introduced into the (inactive) CH bond in the oxidation reaction. In organic chemistry, these are extremely difficult reactions to perform. The OH group is often introduced via a detour, for example, by adding water to a C=C double bond. Selective hydroxylation at specific positions in complex molecules (e.g., bile acids) is problematic because several chemically (almost) equivalent CH bonds exist.

[0006] A method for enzymatically converting lithocholic acid to ursodeoxycholic acid using 7β-hydroxylase is disclosed in International Publication No. 2018 / 227940.

[0007] Schmitz et al. (Microbial Cell Factories 13(1):1-13(2014)) describe the production of 7-hydroxy derivatives of dehydroepiandrosterone (DHEA) or pregnenolone (PREG) by conversion using cytochrome P450 monooxygenase.

[0008] Chinese Patent No. 102002518 discloses the conversion of 3-β-cholesterol acetate to 7-β-hydroxyl-3-β-cholesterol acetate by hydroxylase.

[0009] International Publication No. 2020 / 109776 discloses methods for hydroxylating or dealkylating various organic compounds, particularly those derived from the steroid pregnenolone, with cytochrome P450.

[0010] The object of the present invention is to provide means and methods for hydroxylating steroids, such as bile acids and their derivatives, which have a hydrogen atom at the 7-position and do not have a hydroxyl group at this stage. [Overview of the Initiative] [Means for solving the problem]

[0011] The object of the present invention is achieved by a method for preparing a steroid having general formula (I), [ka] During the ceremony, X1 and X2 are independently H, Cl, F, Br, I, CF3, C1-C6 alkyl group, OH, C1-C6 alkoxy group, CN, NO2, N(R6)2, epoxy group, CHO, or CO2R6 group, where, R6 is -C(O)H, -C(O)CH3, -C(O)CH2CH3, -C(O)(CH2)2CH3, -C(O)CH(CH3)2, -C(O)(CH2)3CH 3, -C(O)CH(CH3)CH2CH3, -C(O)CH2CH2(CH3)2, -C(O)C(CH3)3, -C(O)Ph, -C(O)CH2Ph, R1 and R2 are independently H, OH, OR7, or O, in the formula, R7 is -C(O)H, -C(O)CH3, -C(O)CH2CH3, -C(O)(CH2)2CH3, -C(O)CH(CH3)2, -C(O)(CH2)3CH 3, -C(O)CH(CH3)CH2CH3, -C(O)CH2CH2(CH3)2, -C(O)C(CH3)3, -C(O)Ph, -C(O)CH2Ph, R3 is H, OH, OR8, C1~C 10 Alkyl alkyl groups, C1-C 10 The alkenyl group is -CHO, -C(O)(CH3), -C(O)(CH2OH), -CH(CH3)C(O)CH3, -CH(CH3)((CH2)2CO2R9) or -CH(CH3)((CH2)2CONHR9), in which, R8 is -C(O)H, -C(O)CH3, -C(O)CH2CH3, -C(O)(CH2)2CH3, -C(O)CH(CH3)2, -C(O)(CH2)3CH 3, -C(O)CH(CH3)CH2CH3, -C(O)CH2CH2(CH3)2, -C(O)C(CH3)3, -C(O)Ph or -C(O)CH2Ph, R9 is -CH3, -CH2COOH, -CH2CH3, -CH(CH3)2, -(CH2)2CH3, -(CH2)2SO3H, C(CH3)3, -(CH2)3CH3, -CH(CH3)CH2CH3, -CH2CH2(CH3)2, an aryl group, or an alkylaryl group. R4 is H, OH, or -OR 10 And in the formula, R 10 is -C(O)H, -C(O)CH3, -C(O)CH2CH3, -C(O)(CH2)2CH3, -C(O)CH(CH3)2, -C(O)(CH2)3CH3 , -C(O)CH(CH3)CH2CH3, -C(O)CH2CH2(CH3)2, -C(O)C(CH3)3, -C(O)Ph or -C(O)CH2Ph, R5 is H, CF3, C1-C6 alkyl group, C1-C6 alkenyl group, OH, O, or C1-C6 alkoxy group, where dashed lines indicate any double bond; however, if ring A has a C4-C5 double bond, ring B does not have a double bond; and if X1 and X2 form epoxy groups, ring C does not have a double bond. A 7-deoxysteroid having general formula (II) [ka] The process includes converting 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, wherein the cytochrome P450 enzyme comprises an amino acid sequence that is at least 90%, particularly 100%, identical to the amino acid sequence of SEQ ID NO: 1 or 2.

[0012] Surprisingly, cytochrome P450 and its functional fragments have been shown to be able to hydroxylate steroids such as cholic acid and its derivatives having formula (I) at the 7-position. 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 towards the final product, and thus its yield can be significantly increased. In this case, the regeneration of the redox partner system can preferably occur in the presence of a second system (regeneration system) containing at least one redox enzyme and at least one substrate of the at least one redox enzyme.

Embodiments for Carrying Out the Invention

[0013] Cytochrome P450 and its functional variants that require the oxidation of the reducing equivalent NAD(P)H can surprisingly selectively hydroxylate 7-deoxysteroids such as 7-deoxycholic acid and its derivatives at the 7-position.

[0014] According to the present invention, the cytochrome P450 enzyme comprises an amino acid sequence that is at least 90%, particularly 100% identical to the amino acid sequence of SEQ ID NO: 1 or 2.

[0015] Cytochrome P450 catalyzes the monooxygenase reactions of a number of endogenous and exogenous substrates. They are involved, among other things, in the metabolism of steroids, eicosanoids, fatty acids and bile acids, as well as exogenous substrates such as drugs, pesticides and chemical carcinogens.

[0016] The cytochrome P450 according to the present invention can be used, for example, from bacteria such as actinobacteria, particularly from the genus Streptomyces. In this case, the sequence can be isolated, for example, from genomic DNA or cDNA libraries using known techniques.

[0017] The cytochrome P450s and their functional variants according to the present invention may be present in their original organisms, or isolated from them, or they may be recombinantly expressed or synthetically produced. It is preferable to use recombinant expression polypeptides according to the present invention.

[0018] For example, various established microorganisms such as Escherichia coli (E. coli), Bacillus subtilis, Saccharomyces cerevisiae, or Pichia pastoris can be used for the recombinant expression of the enzyme according to the present invention. Appropriate protocols in this regard are described in detail in the relevant specialized literature or are known to those skilled in the art.

[0019] According to the present invention, the enzyme / polypeptide is preferably used as a recombinantly overexpressed protein in Escherichia coli (E. coli), and the corresponding cell lysate is preferably used without further processing / purification, or after relatively simple processing steps (e.g., centrifugation, precipitation, concentration, or lyophilization). After recombinant overexpression of the enzyme used, Escherichia coli (E. coli) cells can alternatively be used directly in the reaction without cell disintegration, or, for example, after a freeze / thaw cycle. Suitable expression plasmids are known to those skilled in the art and are often commercially available.

[0020] A "functional variant" of cytochrome P450 may be a fragment of cytochrome P450 or a mutant variant, where a fragment of cytochrome P450 can also be called a "functional fragment." A "functional variant" of cytochrome P450 can catalyze the same reactions as the protein from which the variant originates. Whether a variant is functional, that is, whether it catalyzes the same reactions as the protein from which it originates, can be determined by establishing that the variant catalyzes the same reactions. For this purpose, methods described in the prior art or herein have been established, respectively. The conversion rate of substrates by the functional variant according to the present invention may deviate from the conversion rate of the cytochrome P450 from which the variant originates.

[0021] "Derivatives of 7-deoxysteroids" include compounds derived from 7-deoxysteroids that have a wide variety of modifications as defined above.

[0022] According to a preferred embodiment of the present invention, X1, X2, R4 and R5 are H, R1 and R2 are independently H, OH, OR8, or O, in the formula, R8 is -C(O)H, -C(O)CH3, -C(O)CH2CH3, -C(O)(CH2)2CH3, -C(O)CH(CH3)2, -C(O)(CH2)3CH 3, -C(O)CH(CH3)CH2CH3, -C(O)CH2CH2(CH3)2, -C(O)C(CH3)3, -C(O)Ph, -C(O)CH2Ph, R3 is C1~C 10 Alkyl alkyl groups, C1-C 10 The alkylene group is -CH(CH3)((CH2)2CO2R9) or -CH(CH3)((CH2)2CONHR9), and in the formula, R9 is -CH3, -CH2COOH, -CH2CH3, -CH(CH3)2, -(CH2)2CH3, -(CH2)2SO3H, C(CH3)3, -(CH2)3CH3, -CH(CH3)CH2CH3, -CH2CH2(CH3)2, an aryl group, or an alkylaryl group.

[0023] According to a more preferred embodiment of the present invention, the aryl group is selected from the group consisting of a phenyl group, a phenyl group substituted with F, Cl, Br, NO2, or CH3, and heteroaryl groups.

[0024] According to yet another preferred embodiment of the present invention, the alkylaryl group is selected from the group consisting of a benzyl group, a benzyl halide whose halogen is F, Cl, or Br, and a benzyl group substituted with NO2.

[0025] According to a preferred embodiment of the present invention, R1 is OH, R2 is O or OH, R3 is CH(CH3)((CH2)2CO2R5), R4 is H, and R5 is H.

[0026] According to another preferred embodiment of the present invention, the 7-deoxysteroid having general formula (II) is selected from the group consisting of 3α,12α-dihydroxy-5β-colan-24-acid, 3α,12β-dihydroxy-5β-colan-24-acid, 3β,12α-dihydroxy-5β-colan-24-acid, 3β,12β-dihydroxy-5β-colan-24-acid, 3β-hydroxy-12-keto-5β-colan-24-acid, 3-keto,12β-hydroxy-5β-colan-24-acid, 3-keto,12α-hydroxy-5β-colan-24-acid, 3α-hydroxy-5β-colan-24-acid, 3-keto-5β-colan-24-acid, 3β-hydroxy-5β-colan-24-acid and esters of each of these acids.

[0027] The cytochrome P450 enzyme used in accordance with the present invention for the hydroxylation of 7-deoxysteroids having general formula (II) and derivatives thereof to steroids having general formula (I) or derivatives thereof comprises an amino acid sequence that is at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and especially 100%, identical to the amino acid sequence of SEQ ID NO: 1 or 2. Sequence ID 1: [ka] Sequence ID 2: [ka]

[0028] The amino acid sequences of SEQ ID NOs: 1 and 2 are preferably encoded by the nucleic acid sequences of SEQ ID NOs: 3 and 4, and the nucleic acid sequences of SEQ ID NOs: 5 and 6 are optimized for expression in Escherichia coli (E. coli). Sequence ID 3: [ka] Sequence ID 4: [ka] Sequence ID 5: [ka] Sequence ID 6: [ka]

[0029] In this specification, “identical” means that, when superimposed, two or more amino acid sequences may have a certain degree of “identity” (matching amino acid residues at the same position). In this invention, “identity” is defined as the percentage of amino acids in a qualifying amino acid sequence that are identical to the amino acids of the start sequence, i.e., with all variable parameters set to their default values, after, if necessary, alignment and gap introduction of the two sequences, in order to achieve the maximum percentage of sequence identity generated by the “Protein BLAST” program (blastp, Altschul et al., J.Mol.Biol.(1997)215:403-410, http: / / blast.ncbi.nlm.nih.gov / Blast.cgi, commonly referred to herein as “BLAST”). Here, the algorithm "blastp (protein-protein-BLAST)" was used with the following parameters: "expect threshold": 0.05, "word size": 6, matrix: BLOSUM62, "gap cost": "Existence" 11, "Extension" 1, conditional synthesis score matrix adjustment, no filter, and no mask. The percentage (%) value of amino acid sequence identity is determined by dividing the number of matching identical nucleotides by the sequence length for which identity is recorded as a percentage.

[0030] Using the method according to the present invention, a 7-deoxysteroid having general formula (II) or its respective derivative can be converted to a steroid having general formula (I) or its respective derivative using cytochrome P450 or a functional variant thereof, with a cytochrome P450 enzyme containing an amino acid sequence that is at least 90%, particularly 100%, identical to the amino acid sequence of SEQ ID NO: 1 or 2. This conversion occurs in the presence of a redox partner or a redox partner system capable of providing electrons for a hydroxylation reaction.

[0031] According to a preferred embodiment of the present invention, at least one redox partner system is (i) Ferredoxin, ferredoxin reductase and NAD(P)H, (ii) Cytochrome P450 reductase and NAD(P)H or (iii) NAD(P)H Includes.

[0032] The redox partner system used in accordance with the present invention may include ferredoxin, ferredoxin reductase and NAD(P)H, cytochrome P450 reductase and NAD(P)H, or NAD(P)H alone, with a redox partner system containing ferredoxin, ferredoxin reductase and NAD(P)H being particularly preferred.

[0033] Therefore, in order to carry out the redox reaction of cytochrome P450 or each of its functional variants according to the present invention, it is advantageous to use at least the redox cofactors NAD+ / NADH and / or NADP+ / NADPH in the method according to the present invention. In this regard, NAD+ represents the oxidized form of nicotinamide adenine dinucleotide, NADH represents the reduced form, NADP+ represents the oxidized form of nicotinamide adenine dinucleotide phosphate, and NADPH represents the reduced form.

[0034] The concentration of the redox cofactors NAD(P)+ and / or NAD(P)H in the reaction mixture is preferably 0.001 mM to 10 mM, more preferably 0.05 mM to 1 mM.

[0035] Particularly preferably, ferredoxin is used as the redox partner, which can be regenerated in the presence of NAD(P)+ and at least one ferredoxin reductase. According to a preferred embodiment of the present invention, at least one ferredoxin is selected from the group consisting of adrenodoxin, ptydaredoxin and flavodoxin, and optionally, a combination thereof can also be used.

[0036] Possible pairs of redox partners preferably include ptydaredoxin and ptydaredoxin reductase derived from Pseudomonas putida. Furthermore, those skilled in the art can identify additional ferredoxin proteins and ferredoxin reductases that are potential redox partners of cytochrome P450 according to the present invention. Suitability as a redox partner can be verified by functional assays, for example, as described in Examples 3-5. The ptydaredoxin and / or ptydaredoxin reductase used in these examples can each be replaced with possible alternative proteins or enzymes. If sufficient formation of the desired product (e.g., ursocholic acid) is observed, the tested redox partner can be considered a functional substitute for ptydaredoxin and / or ptydaredoxin reductase.

[0037] According to a particularly preferred embodiment of the present invention, the ferredoxin used in the method according to the present invention comprises an amino acid sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and especially 100% identical to the amino acid sequence of SEQ ID NO: 7, where X is either a methionine residue or not an amino acid. Sequence ID 7: [ka]

[0038] According to a preferred embodiment of the present invention, at least one ferredoxin reductase is selected from the group consisting of flavodoxin reductase and ptydaledoxin reductase.

[0039] The ferredoxin oxidized during the hydroxylation reaction according to the present invention can be reduced with the help of ferredoxin reductase and NAD(P)H. As a result, reduced ferredoxin is provided again, or each is regenerated while consuming NAD(P)H for further hydroxylation reactions of the substrate according to the present invention. The ferredoxin reductase may be flavodoxin reductase and / or ptydaledoxin reductase.

[0040] According to a more preferred embodiment of the present invention, the ferredoxin reductase used in the method according to the present invention comprises an amino acid sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and especially 100% identical to the amino acid sequence of SEQ ID NO: 8. Sequence ID 8: [ka]

[0041] The amino acid sequences of SEQ ID NOs. 7 and 8 are preferably encoded by the nucleic acid sequences of SEQ ID NOs. 9 and 10, respectively, and the nucleic acid sequences of SEQ ID NOs. 11 and 12 are optimized for expression in Escherichia coli (E. coli). Sequence ID 9: (ATG) 0又は1 [ka] Sequence ID 10: [ka] Sequence ID 11: (ATG) 0又は1 [ka] Sequence ID 12: [ka]

[0042] The expression of cytochrome P450 and ferredoxin and ferredoxin reductase according to the present invention in bacteria, particularly Escherichia coli (E. coli), is particularly advantageous when using nucleic acids having the nucleic acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11 and / or SEQ ID NO: 12. Therefore, further embodiments of the present invention relate to nucleic acids (DNA and / or RNA) having 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, particularly Escherichia coli (E. coli) cells, comprising at least one of these sequences.

[0043] It has been shown that co-expression of ferredoxin and ferredoxin reductase with cytochrome P450 in producing strains (e.g., Escherichia coli strains) is advantageous. Ferredoxin, ferredoxin reductase, and cytochrome P450 can also be expressed separately from each other. Co-expression of ferredoxin and cytochrome P450 or ferredoxin reductase and cytochrome P450 is also advantageous. Ideally, co-expression of the three proteins or each enzyme under the same promoter can establish an ideal balance between the enzymes, which has a particularly advantageous effect on the enzymatic conversion of substrates.

[0044] According to a preferred embodiment of the present invention, 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), preferably selected from the group consisting of alcohol dehydrogenase, hydroxysteroid dehydrogenase, phosphite dehydrogenase and sugar dehydrogenase.

[0045] For the redox partner system used in the method according to the invention, in particular for the cofactor (NADH / NAD + and / or NADPH / NADP + ) to be regenerated, during the conversion of a steroid having the general formula (I) to a 7-deoxysteroid having the general formula (II), it is advantageous to add to the reaction mixture a redox partner system, preferably a system for regenerating the oxidoreductase, so that the conversion reaction proceeds towards the product. The oxidoreductase converts the substrate by reduction and oxidation, and during these reactions, NADH is oxidized to NAD + , NADPH is oxidized to NADP + , or NAD+ is reduced to NADH and NADP + is reduced to NADPH, respectively. Therefore, the system for regenerating the redox partner system preferably contains at least one oxidoreductase and at least one substrate of the at least one oxidoreductase.

[0046] The oxidoreductase used in the method according to the invention is preferably an alcohol and / or sugar dehydrogenase.

[0047] According to a more preferred embodiment of the present invention, the oxidoreductase is selected from the group consisting of glucose dehydrogenase, glucose-6-phosphate dehydrogenase, arabinose dehydrogenase, xylose dehydrogenase, sorbitol dehydrogenase, xylitol dehydrogenase, 12α-hydroxysteroid dehydrogenase, 7α-hydroxysteroid dehydrogenase, 20α-hydroxysteroid dehydrogenase, 17β-hydroxysteroid dehydrogenase, 17α-hydroxysteroid dehydrogenase, 3α-hydroxysteroid dehydrogenase, 3β-hydroxy-δ5 dehydrogenase, 11β-hydroxysteroid dehydrogenase, and formate dehydrogenase.

[0048] The use of one or more of the aforementioned oxidoreductases is particularly advantageous for reusing cofactors used in the conversion reaction.

[0049] To achieve a high conversion of the substrate to the product, it has been shown to be particularly advantageous to add arabinose dehydrogenase, sorbitol dehydrogenase, and / or xylitol dehydrogenase to the reaction mixture.

[0050] The reaction mixture may contain at least one oxidoreductase and one hydroxylase. Adding a combination of two or more oxidoreductases to the reaction mixture is particularly advantageous, and combinations of 12α-hydroxysteroid dehydrogenase and 7α-hydroxysteroid dehydrogenase, or 12α-hydroxysteroid dehydrogenase, 7α-hydroxysteroid dehydrogenase and NAD(P)H oxidase respectively, or NADH-dependent alcohol dehydrogenase and hydroxylase respectively, or NADPH-dependent alcohol dehydrogenase and hydroxylase respectively are particularly well suited for the simultaneous oxidation and hydroxylation of substrate mixtures, such as naturally occurring mixtures of cholic acid.

[0051] In order to catalyze the oxidation or reduction reaction of the cofactors in the reaction mixture of the method according to the present invention, it is necessary to provide at least one substrate for the oxidoreductase present therein. Accordingly, the reaction mixture contains at least one substrate for at least one oxidoreductase selected from the group consisting of arabinose, xylose, glucose, sorbitol, xylitol, coran-24-acid, 3α,12α-dihydroxycoran-24-acid-2,3-butanediol, acetoin, 2-propanol, glutamate, ethanol, phosphonate, phosphine, nitrite, 4-methyl-2-pentanol, 2-butanol, 2-octanol, cyclohexanol, ethanediol, 1,2-propanediol, 1-propanol, 1-butanol, 3-hydroxybutanoate, and formate. According to a preferred embodiment of the present invention, the method according to the present invention is carried out at a temperature of 10°C to 40°C, preferably 15°C to 38°C, more preferably 20°C to 30°C, and more preferably 22°C to 26°C. According to the present invention, the enzymatic activity of cytochrome P450 for the reaction according to the present invention is particularly high in this region.

[0052] According to a more preferred embodiment of the present invention, the method according to the present invention is carried out at a pH of 6.5 to 8.5, preferably 7 to 8, and more preferably 7.2 to 7.8. At this pH value, the enzymatic activity of cytochrome P450 is highest to enable proper substrate conversion.

[0053] The hydroxylation of deoxysteroids or their respective derivatives can be performed regioselectively at the 7th position of the steroid skeleton. In this way, the 7β-hydroxyl group can be stereoselectively introduced, particularly to produce ursocholic acid and / or ursocholic acid derivatives.

[0054] In preferred embodiments, the method according to the present invention is carried out in the presence of at least one organic solvent. Preferably, a single organic solvent is used so as to provide a single-phase system. According to the present invention, it is also possible to use a mixture of two or more organic solvents that are miscible with respect to each other so as to provide a single-phase system. The organic solvent may be protic or aprotic, and an aprotic solvent is preferred.

[0055] Surprisingly, it has been found that the presence of organic solvents, particularly aprotic organic solvents, can significantly increase the conversion of 7-deoxysteroids of general formula (II) to steroids of general formula (I) by the method according to the present invention. Furthermore, surprisingly, carrying out the method according to the present invention in the presence of organic solvents enables the regeneration of the redox partner system.

[0056] For example, alcohols, ethers, esters, glycols, ketones, amides, sulfoxides, organic acids, cycloalkanes, aromatics, and chlorinated hydrocarbons can be used as organic solvents. Examples of suitable organic solvents include methanol, ethanol, isopropanol, 2-butanol, 4-methyl-2-pentanol (methyl isobutyl alcohol, MIBA), diethyl ether (Et2O), diisopropyl ether (iPr2O), dioxane, tetrahydrofuran (THF), 2-methyltetrahydrofuran (Me-THF), ethyl acetate, ethylene glycol, methyl isobutyl ketone (MIBK), 2-butanone, acetone, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), cyclohexane, toluene, trichloromethane (CHCl3), dichloromethane (CH2Cl2), hexane, or mixtures thereof. Suitable mixtures include, for example, mixtures of hexane and ethyl acetate or isopropanol, and mixtures of trichloromethane and phenol. The present invention is not limited to the above list of exemplary solvents.

[0057] As the organic solvent, an aprotic organic solvent is preferred, and a solvent selected from the group consisting of dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and dimethylacetamide (DMA) is particularly preferred.

[0058] According to the present invention, the amount of organic solvent is selected such that the compound of general formula (II) is completely dissolved and the enzyme activity is preserved. Preferably, the compound of formula (II), such as litcholic acid, is dissolved in the organic solvent to its solubility limit. In a preferred embodiment, the enzyme substrate is placed in the organic solvent.

[0059] In the method according to the present invention, the isolation of the product can be carried out by different methods. For example, the product can be extracted from the reaction mixture with a suitable organic solvent. Depending on the substrate, such solvents are described in the literature. According to the present invention, cholic acid and its derivatives can optionally be isolated from the reaction mixture with, for example, ethyl acetate after acidifying the reaction mixture with, for example, HCl. A special case is when bile acids are present in aqueous solution in the form of a salt, for example, a sodium salt.

[0060] In this case, precipitation of the product can be carried out by acidifying the reaction mixture. For this purpose, for example, a sufficient amount of HCl or dilute HCl can be added to the reaction mixture. For example, if a pH value of 1 to 4, preferably 2 to 3, is achieved in the process, the product will exist mainly in the form of a suspension. The product can then be removed from the reaction mixture by common methods such as filtration or centrifugation. Chromatographic methods such as affinity chromatography or ion exchange chromatography are another alternative method that can be used, for example, to isolate the product. Furthermore, the product can be obtained, for example, by evaporating the reaction solvent.

[0061] Alternatively, in the method according to the present invention, the product may also remain in the reaction mixture after the reaction to carry out further reactions, for example, and optionally to isolate the final product upon completion of those reactions. One or more substrates for the method according to the present invention may also be prepared in the same reaction batch by a previous or parallel reaction.

[0062] Further aspects of the present invention relate to nucleic acid constructs comprising a nucleic acid molecule encoding a cytochrome P450 enzyme as defined above, with at least one nucleic acid molecule encoding a polypeptide selected from the group consisting of ferredoxin, ferredoxin reductase, and oxidoreductase directly or via a spacer bound to its 3' and / or 5' ends.

[0063] The nucleic acid constructs according to the present invention are particularly suitable for the production of the initially defined steroids. By expressing a cytochrome P450 enzyme and at least one protein selected from the group consisting of ferredoxin, ferredoxin reductase, and oxidoreductase starting from the nucleic acid construct, it becomes possible to produce these enzymes or each of these proteins in amounts necessary for the efficient implementation of the method according to the present invention. It is particularly advantageous if all of these proteins are expressed under the control of the same promoter on the nucleic acid construct according to the present invention. A further aspect of the present invention is a vector comprising the nucleic acid construct according to the present invention.

[0064] Nucleic acid molecules encoding cytochrome P450 enzymes can be conjugated directly to or via spacers or spacer sequences to further nucleic acid molecules encoding enzymes or proteins, respectively, that can be used in the methods according to the present invention. The advantage of such constructs is that they enable the expression of enzymes and proteins used in the methods according to the present invention, particularly cytochrome P450 and ferredoxin and / or ferredoxin reductase, in comparable amounts.

[0065] As used herein, “spacers” or “spacer sequences” are nucleic acid sequences that do not contain stop codons or other functional motifs. Spacers or spacer sequences may, if necessary, act as distance holders between two ORFs to improve the transcription of those ORFs.

[0066] According to a preferred embodiment of the present invention, the cytochrome P450 enzyme is encoded by a nucleic acid that is at least 90%, and particularly 100%, identical to the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0067] According to a more preferred embodiment of the present invention, ferredoxin comprises the amino acid sequence of SEQ ID NO: 7.

[0068] According to a particularly preferred embodiment of the present invention, the ferredoxin reductase is adrenodoxin reductase, preferably ptydaledoxin reductase.

[0069] Ferredoxin reductase preferably contains the amino acid sequence of SEQ ID NO: 8.

[0070] Another aspect of the present invention relates to a vector comprising a nucleic acid construct according to the present invention.

[0071] The vector according to the present invention may be a cloning or expression vector, and may have a portion appropriate to enable, for example, the transcription of an ORF, depending on the organism into which the vector is introduced.

[0072] A further aspect of the present invention relates to a host cell comprising a nucleic acid construct according to the present invention.

[0073] The host cells according to the present invention can be recombinantly introduced and used to clone or express ORFs located on nucleic acid constructs.

[0074] Such host cell lysates can be used in the method according to the present invention, provided that the host cells express at least one of the enzymes or proteins required by the method according to the present invention, either intracellularly or extracellularly. It is preferable to use a 7-deoxysteroid having general formula (II) or a derivative thereof to contact the cell suspension or cells in the culture supernatant and / or lysate of the host cell according to the present invention. Accordingly, according to a preferred embodiment of the present invention, a 7-deoxysteroid having general formula (II) or a derivative thereof is used to contact the culture supernatant and / or lysate of at least one host cell capable of expressing at least one ferredoxin, at least one ferredoxin reductase and / or at least one oxidoreductase.

[0075] example The present invention will be further illustrated using the following examples, but it is not limited thereto.

[0076] Example 1: Testing bacterial strains From the German collection of microorganisms and cell cultures (DSMZ [Deutsche Stammsammlung fur Mikroorganismen und Zellkulturen]), the following strains were obtained: Saccharothrix longispora (DSM-43749), Catellatospora citrae (DSM-44097), Streptomyces hygroscopicus subsp. hygroscopicus (DSM-40578), and Asanoa ferruginea (DSM-44099). The strains were cultured under standard conditions recommended by DSMZ. As soon as the culture growth reached a visible turbidity, deoxycholic acid (0.5 mM) was added, and the culture was further cultured for up to 72 hours. After centrifugation, the culture supernatant was extracted with ethyl acetate and analyzed by HPLC and GC / MS. HPLC chromatograms of the reaction with Streptomyces hygroscopicus showed peaks corresponding to the retention time of ursocholic acid. GC / MS analysis indicated that the potential ursocholic acid peak originated from a bile acid with three hydroxyl groups. Testing of other strains did not show a 7-hydroxylated product of deoxycholic acid.

[0077] Example 2: Genome sequencing and annotation of the P450 gene Streptomyces hygroscopicus subsp. hygroscopicus (DSM-40578) was cultured according to DSMZ standards, and its genomic DNA was isolated (Kieser et al. (2000), Practical Streptomyces genetics (Norwich: John Innes Foundation)). The genome was sequenced using Illumina MiSeq and assembled based on the genome of the known Streptomyces rapamycinicus (Microsynth GmbH, Switzerland). Forty-two P450 genes were identified by homology comparison.

[0078] Example 3: Cloning of the expression system Using restriction enzyme XhoI, the following constructs containing the coding regions for ptydaredoxin reductase (PtR) and ptydaredoxin (Ptx) were cloned into plasmid pJ411 (DNA2.0).

[0079] Synthetic DNA (Life Technologies): 5', XhoI interface, HindIII interface, approximately 50 bp spacer DNA, ribosome binding site (rbs), ORF (open reading frame) ptydaredoxin reductase (PtR), approximately 50 bp spacer DNA, rbs, ORF ptydaredoxin (Ptx), XhoI interface, 3'.

[0080] The results of the cloning process were confirmed by restriction enzyme digestion and DNA sequencing.

[0081] Next, using restriction enzymes NdeI and HindIII, one ORF encoding the P450 enzyme identified in Example 2 was cloned into the synthetic DNA and plasmid, respectively (Life Technologies). These results were again verified by restriction enzyme digestion and DNA sequencing. The expression vector and redox partner used in this example constitute only one method for expressing the cytochrome P450 enzyme according to the present invention, and this method is selected as an example.

[0082] Expression plasmids produced using the identified P450 candidates (see Example 2) can be used to co-express each P450 protein together with ptydaredoxin reductase and ptydaredoxin. The three ORFs of each expression plasmid are expressed under the control of a T7 promoter on a common mRNA, but are expressed as separate polypeptides.

[0083] Example 4: Expression of P450 / Ptx / PtR Following genome sequencing of Streptomyces hygroscopicus subsp. hygroscopicus, 42 P450 sequences were considered as potential candidates for deoxycholic acid-7-hydroxylase. To identify the enzyme of interest, candidate ORFs were cloned into the expression system described in Example 3 and then cloned into the pJ411 (DNA2.0) expression vector without the coding regions for ptydaredoxin reductase (PtR) and ptydaredoxin (Ptx). The following protocol was used for expression.

[0084] TB-P450 expression medium: Terrific broth (TB) medium +50μg / ml Kanamycin +0.5 mM 5-aminolevulinic acid (from 100 × parent solution) +1 mM thiamine (from 100 × parent solution) +1 mM MgCl2 + 2.5 mM ammonium sulfate + 50 μM FeCl3 (from 100 × parent solution) +0.5mM IPTG (from 1M parent solution) (Each additive was sterilized and filtered to 0.2 μm.) P450 lysis buffer: 100mM Tris pH7.5 20% (v / v) Glycerin 1 mg / ml lysozyme

[0085] The P450 candidate constructs to be tested were transformed into Escherichia coli (E. coli) strain BL21(DE3). Overnight cultures were seeded from single colonies (LB (lysogenic broth) + kanamycin). The following day, these were seeded into 1:100 expression cultures (150 ml of TB (terrific broth)-P450 expression medium) and first shaken in a baffled flask (1 L) at 37°C for 3 hours. Then the temperature was reduced to 24°C and shaken for a further 22 hours. The cultures were collected by centrifugation at 5000 g for 10 minutes, washed once with 0.9% (w / v) NaCl, and the pellet was frozen at -80°C. The cell pellet was thawed, weighed, resuspended in an equal volume of P450 lysis buffer, incubated on ice for 1 hour, and then digested using an ultrasonic device. Centrifugation (30 minutes, 21000 g) was performed, and the supernatant was used for the test reaction.

[0086] Example 5: Testing of P450 candidates for DA hydroxylation Reaction mixture: 10-80 μl of 100 mM NADH (oxidation-reduction cofactor) 250 μl of 1 M Tris-HCl, pH 7.5 17.5 μl of glycerin (50%) 100 μl of 50 mM deoxycholic acid solution, pH 8.5 (final 10 mM) 50 μl of E. coli lysate P450 / PtR / Ptx (see Example 4) 17.5-87.5 μl dH2O

[0087] The reaction mixture was placed in a 1.5 ml screw-top vial and capped with aluminum foil. Several holes were made in the foil. This was gently shaken at 24°C for 18 hours. A 200 μl reaction batch was diluted with 600 μl acetonitrile / 5 μl H3PO4 (50%) and incubated at 55°C for 15 minutes. The sample was then centrifuged at 20817rcf for 5 minutes and subjected to HPLC / DAD (e.g., Agilent 1200 series, column: Merck Purospher STAR RP-18e 125×4 mm, 5 μm). Analysis was performed using a flow rate of 1.5 ml / min and a gradient of H2O + H3PO4 (pH=2.6) / acetonitrile. One of the candidates investigated ("P450_c866") was able to hydroxylate deoxycholic acid to ursocholic acid. The deoxycholic acid used was converted in this process (see table below). The identity of the product ursocholic acid was verified by GC / MS analysis and 2D NMR. [Table 1]

[0088] In this example, the redox cofactor (NADH) is oxidized by the P450 / Ptx / PtR reaction.

[0089] Example 6: Testing of P450 candidates for DA hydroxylation via cofactor recycling of arabinose dehydrogenase Reaction mixture: 65 μl of 100 mM NADH (final concentration 0.5 mM) 1 ml of 1 M Tris-HCl pH 7.5 + 20% (v / v) glycerin 130 μl of 50 mM deoxycholic acid solution, pH 8.5 (final 0.5 mM) 6.5 μl of chloramphenicol solution (final concentration 20 μg / ml) 100 μl of L-arabinose dehydrogenase (From Burkholderia vietnamiensis recombinantly expressed in Escherichia coli (E. coli), 400 U / ml) 98 mg of L-arabinose (final 50 mM) 2.0 ml of E. coli lysate P450 / PtR / Ptx (see Example 4) 9.6 ml dH2O

[0090] The reaction mixture was placed in a 50 ml baffleless Erlenmeyer flask and sealed with aluminum foil. Several holes were made in the film. This was gently shaken at 24°C for 16 hours. The substance present in the supernatant was extracted with ethyl acetate and evaporated. This was dissolved in a smaller amount of HPLC eluent (methanol / acetonitrile / H2O+H3PO4 (pH=3.0), 40:30:33). Subsequently, the sample was analyzed using HPLC / RID (e.g., Agilent 1200 series, column: Agilent ZORBAX Eclipse XDB-C18 4.6×150 mm, 5 μm, flow rate: 0.8 ml / min). One of the candidates investigated, "P450_c866," was able to hydroxylate deoxycholic acid to ursocholic acid. The deoxycholic acid used was almost completely converted (over 95%) in this process. The identity of the product ursocholic acid was verified by GC / MS analysis and 2D NMR (data not shown).

[0091] In this example, the redox cofactor (NADH) is oxidized by the P450 / Ptx / PtR reaction. The redox cofactor is then reduced to its original state by cofactor regeneration (in this case, for example, using sugar dehydrogenase or arabinose dehydrogenase) (in this case, arabinose is oxidized to arabinolactone / aravonic acid). This makes it possible to use a quasi-stoichiometric amount of the redox cofactor.

[0092] Example 7: Example: Conversion dependent on cofactor concentration transformation by cofactor recycling Reaction mixture: 10 μl of 10 mM NAD+ 250 μl of 100 mM TEA, pH 8.2 25 μl of glycerin (50%) 100 μl of 50 mM deoxycholic acid solution, pH 8.0 (final 10 mM) 6.75 mg of cells (wW) as a 22.5% suspension in 100 mM TEA pH 8.0 and 25% glycerin. 5 μl catalase (bovine, Sigma, 4 mg / ml) 1.7 units of xylitol / sorbitol dehydrogenase 25 μl of 2M sorbitol (final concentration 100 mM) 70.8 μl dH2O

[0093] The reaction mixture was placed in a 1.5 ml screw-top bottle and covered with aluminum foil. Several holes were made in the foil. This was then gently shaken at 24°C for 18 hours.

[0094] NADH recovery involves sorbitol and NAD + This was carried out in the presence of sorbitol or xylitol dehydrogenase, respectively.

[0095] A 200 μl reaction batch was diluted with 600 μl acetonitrile / 5 μl H3PO4 (50%) and incubated at 55°C for 15 minutes. The sample was then centrifuged at 20817rcf for 5 minutes and analyzed using HPLC / DAD (e.g., Agilent 1200 series, columns: Merck Purospher STAR RP-18e 125×4 mm or Agilent Zorbax XDB-C 8 mm 150×4.6 mm, 3.5 μm, 5 μm, flow rate: 1.5 ml / min, gradient H2O+H3PO4 (pH=2.6) / acetonitrile).

[0096] The deoxycholic acid used was quantitatively converted (100% conversion) to ursocholic acid under the above conditions. The identity of the product ursocholic acid was verified by GC / MS analysis and 2D NMR.

[0097] In this example, the cofactor recycling system is sorbitol / xylitol dehydrogenase / sorbitol / NAD + The redox cofactor (NADH) obtained by this process is used in the hydroxylation reaction. However, other systems for cofactor recycling can also be used (see the table below). [Table 2]

[0098] Example 8: Quantitative LCA (Lithocholic Acid) Conversion Reaction mixture: 10 μl of 10 mM NAD+ 250 μl of 100 mM TEA, pH 8.2 10 mM (final) lithocholic acid 9.2 mg of cells (wW) as a 22.5% suspension in 100 mM TEA pH 8.0 and 25% glycerin. 5 μl catalase (bovine, Sigma, 4 mg / ml) 1.7 units of xylitol / sorbitol dehydrogenase 25 μl of 2M sorbitol (final concentration 100 mM) 176 μl dH2O

[0099] The reaction mixture was placed in a 1.5 ml screw-top bottle and covered with aluminum foil. Several holes were made in the foil. This was then gently shaken at 24°C for 18 hours.

[0100] NADH recovery involves sorbitol and NAD + This was carried out in the presence of sorbitol or xylitol dehydrogenase, respectively.

[0101] A 200 μl reaction batch was diluted with 600 μl acetonitrile / 5 μl H3PO4 (50%) and incubated at 55°C for 15 minutes. The sample was then centrifuged at 20817rcf for 5 minutes and analyzed using HPLC / DAD (e.g., Agilent 1200 series, columns: Merck Purospher STAR RP-18e 125×4 mm or Agilent Zorbax XDB-C 8 mm 150×4.6 mm, 3.5 μm, 5 μm, flow rate: 1.5 ml / min, gradient H2O+H3PO4 (pH=2.6) / acetonitrile).

[0102] Under the above conditions, only ursodeoxycholic acid was detected after conversion.

[0103] Example 9: Conversion of LCA (lithocholic acid) to ursodeoxycholic acid in the presence of an organic solvent First, the solubility limits of LCA and UDCA in various organic solvents were measured. For this purpose, 10 mg or 100 mg of LCA or UDCA, respectively, were placed in a 15 mL flask. 100 μL of organic solvent was added at a time, and the mixture was treated by shaking in a vortex shaker and, if necessary, in an ultrasonic bath. The presence or absence of a clear solution was visually assessed.

[0104] The table below summarizes the determined solubility limits for the analyzed solvents. [Table 3]

[0105] The conversion of LCA to UDCA in the presence of a solvent was measured as shown in the table below. [Table 4]

[0106] Example 10: Conversion of lithocholic acid to ursodeoxycholic acid with increased substrate concentration in the presence of an aprotic solvent. Reaction mixture: 10 μl of 10 mM NAD+ 250 μl of 200 mM TEA pH 8.4 containing 10.8% glycerin. 25 μl of 500 mM lithocholic acid in DMF 30 mg of cells (wW) as a 30% suspension in 100 mM TEA pH 9.0 5 μl catalase (bovine, Sigma, 4 mg / ml) 1.7 units of xylitol / sorbitol dehydrogenase 25 μl of 2M sorbitol (final concentration 100 mM) 73 μl dH2O

[0107] The reaction mixture was placed in a 1.5 ml screw-top bottle and covered with aluminum foil. Several holes were made in the foil. This was then gently shaken at 24°C for 18 hours.

[0108] NADH recovery involves sorbitol and NAD + This was carried out in the presence of sorbitol or xylitol dehydrogenase, respectively.

[0109] The reaction batch was completely evaporated in a stream of air and redissolved in 1.1 ml of IPA + 0.5% TFA. The sample was then centrifuged at 20817rcf for 5 minutes, and the supernatant was analyzed by HPLC / RID (e.g., Agilent 1200 series, column: Phenomenex Luna® Silica 100 Å, 250 × 4.6 mm, 5 μm, flow rate: 1.0 ml / min, n-hexane / IPA 4:1 + 0.05% TFA homogeneous concentration).

[0110] Under the above conditions, 70% ursodeoxycholic acid was detected after conversion. The present invention includes the following preferred embodiments. (1) A method for preparing a steroid having general formula (I), [ka] During the ceremony, X 1 and X 2 These are independently H, Cl, F, Br, I, and CF. 3 、C 1 ~C 6 Alkyl alkyl group, OH, C 1 ~C 6 Alkoxy group, CN, NO 2 , N(R 6 ) 2 , epoxy group, CHO or CO 2 R 6 It is the basis, and in the formula, R 6 -C(O)H, -C(O)CH 3 ,-C(O)CH 2 CH 3 -C(O)(CH 2 ) 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)CH 2 CH(CH 3 ) 2 , -C(O)C(CH 3 ) 3 -C(O)Ph, or -C(O)CH 2 Ph is, R 1 and R 2 These are independently H, OH, OR 7 or O, in the formula, R 7 -C(O)H, -C(O)CH 3 ,-C(O)CH 2 CH 3 -C(O)(CH 2 ) 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)CH 2 CH(CH 3 ) 2 , -C(O)C(CH 3 ) 3 -C(O)Ph, or -C(O)CH 2 Ph is, R 3 H, OH, OR 8 、C 1 ~C 10 Alkyl alkyl group, C 1 ~C 10 Alkenyl group, -CHO, -C(O)(CH) 3 ), -C(O)(CH 2 OH), -CH(CH 3 )C(O)CH 3 , -CH(CH 3 )((CH 2 ) 2 CO 2 R 9 ) or -CH(CH 3 )((CH 2 ) 2 CONHR 9 ) and in the formula, R 8 -C(O)H, -C(O)CH 3 ,-C(O)CH 2 CH 3 -C(O)(CH 2) 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)CH 2 CH(CH 3 ) 2 , -C(O)C(CH 3 ) 3 -C(O)Ph or -C(O)CH 2 Ph is, R 9 is, -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 CH 3 , -CH(CH 3 )CH 2 CH 3 ,-CH 2 CH(CH 3 ) 2 , an aryl group or an alkylaryl group, R 4 is H, OH, or -OR 10 And in the formula, R 10 -C(O)H, -C(O)CH 3 ,-C(O)CH 2 CH 3 -C(O)(CH 2 ) 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)CH 2 CH(CH 3 ) 2 , -C(O)C(CH 3 ) 3 -C(O)Ph or -C(O)CH 2 Ph is, R 5 H, CF 3 、C 1 ~C 6 Alkyl alkyl group, C 1 ~C 6 Alkenyl group, OH, O, or C 1 ~C 6 It is an alkoxy group, and in the formula, the dashed line indicates any double bond, however, if ring A has a C4-C5 double bond, ring B does not have a double bond, and X 1 and X 2 When forming an epoxy group, the C ring does not have a double bond. A 7-deoxysteroid having general formula (II)

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Sequence Free-Text

[0111] Sequence Listing 5 <223>Optimized for E. coli codons, SEQ ID NO: 3 Sequence Listing 6 <223>Optimized for E. coli codons, SEQ ID NO: 4 Sequence Listing 7 <223>Xaa is methionine or no amino acid Sequence Listing 9 <223>n is A or no nucleotide Sequence Listing 9 <223>n is T or no nucleotide Sequence Listing 9 <223>n is G or no nucleotide Sequence Listing 11 <223>Optimized for E. coli codons, SEQ ID NO: 9 Sequence Listing 11 <223>n is A or no nucleotide Sequence Listing 11 <223>n is T or no nucleotide Sequence Listing 11 <223>n is G or no nucleotide Sequence Listing 12 <223> E. coli codon optimized for sequence number 10

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