21-Hydroxylation of steroids

Abstract

To provide a method for 21-hydroxylation of steroids in cells.SOLUTION: A method for hydroxylation of carbon atom 21 of a steroid includes the steps of: a) providing a cell that expresses (i) a heterologous CYP21A2 protein or a functional variant thereof, which has at least 20% of ability of unaltered heterologous CYP21A2 protein to 21-hydroxylate a steroid(ii) a heterologous electron transfer system selected from the group consisting of a combination of a flavodoxin reductase (Fpr) and an adrenodoxin, a combination of the Fpr and a ferredoxin domain (etpfd) of an electron transfer domain, a combination of an adrenodoxin reductase homolog (Arh) and the adrenodoxin, a combination of the Arh and the etpfd, and (iii) one or more kinds of chaperones facilitating folding of CYP21A2; and b) adding the steroid to the cell.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates generally to the field of steroid hydroxylation. More particularly, the present invention relates to a method for 21-hydroxylation of steroids in cells. The present invention also relates to cells expressing an enzyme that performs steroid 21-hydroxylation, i.e., a steroid 21-hydroxylase, an expression vector comprising a nucleic acid encoding a steroid 21-hydroxylase, and a kit for carrying out the method for 21-hydroxylation of steroids in cells. [Background technology]

[0002] Synthetic glucocorticoids are derivatives of the naturally occurring stress hormone cortisol and play an important role in the pharmaceutical industry due to their anti-inflammatory and immunosuppressant effects, and synthetic molecules are often more effective than cortisol. Summary of the Invention [Problem to be solved by the invention]

[0003] Currently, the synthesis of some pharma- ceutically active steroids consists of lengthy, multi-step chemical syntheses, including the 21-hydroxylation of their precursors (see Figure 1 for an example). This hydroxylation is also difficult for some synthetic chemists, since chemical oxidants are not selective for the 21-position. For this reason, it is necessary to protect other functional groups to avoid their oxidation and to direct the hydroxylation reaction to the 21-position. Furthermore, this synthesis is not environmentally friendly, due to the use of reagents such as iodine. Therefore, cheap and sustainable production of pharma-ceutically active steroids is highly desirable to meet the high demand for these important drugs. [Means for solving the problem]

[0004] The present inventors address this problem by developing a whole-cell biotransformation of steroids in a single-step synthesis by the enzyme CYP21A2. The enzyme CYP21A2 is a member of the protein family of cytochrome P450 monooxygenases and is capable of highly selective hydroxylation of steroids at position 21 of the steroid backbone (see FIG. 2 for a scheme of the method). CYP21A2 is a mammalian membrane-anchored enzyme that is located in the endoplasmic reticulum and plays a key role in steroid hormone biosynthesis. The present inventors show that the biocatalytic system of the present invention is a promising candidate to replace established chemical synthesis. The present inventors show in particular that steroids can be modified in a single hydroxylation step leading to one desired product. This is time-saving, environmentally friendly (no by-products were observed) and facilitates downstream processing. Additionally and advantageously, the whole-cell steroidogenesis according to the present invention does not require purification of the enzyme, does not require the addition of expensive redox equivalents such as NADPH, since the enzyme remains stable in the host and the cells themselves serve as donors.

[0005] Before the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methods, protocols, and reagents described herein, since the methods, protocols, and reagents may vary. It should also be understood that the terms used herein are merely for the purpose of describing specific embodiments, and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0006] Terms used herein are preferably defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and Kolbl, H. (eds.) (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0007] Several documents are cited in the body of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0008] The components of the present invention are described below. Although these components are listed with specific embodiments, it should be understood that these components may be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments should not be interpreted as limiting the present invention to only the embodiments explicitly described. This description should be understood as supporting and encompassing embodiments that combine the explicitly described embodiments with any number of the disclosed components and / or preferred components. Furthermore, unless otherwise indicated by the context, any permutation and combination of all components described in this application should be considered to be disclosed by the description of this application.

[0009] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" should be understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integers or steps or group of integers or steps. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0010] In a first aspect, the present invention provides a method for hydroxylating carbon atom 21 of a steroid, comprising the steps of: a) (i) a heterologous CYP21A2 protein or a functional variant thereof; (ii) at least one heterologous electron transport system capable of transferring electrons to CYP21A2; and (iii) one or more chaperones that facilitate folding of CYP21A2 Providing a cell expressing b) adding steroids to the cells The present invention relates to a method comprising the steps of:

[0011] Steroids are a class of organic compounds that contain four cycloalkane rings linked together in a characteristic arrangement (shown below). The steroid nucleus is composed of 17 carbon atoms bonded together in the form of four fused rings: three cyclohexane rings (designated rings A, B, and C) and one cyclopentane ring (ring D). Steroids differ by the functional groups attached to this four-ring nucleus and by the oxidation states of the rings.

[0012] Hydroxylation of carbon atom 21 of a steroid is the addition of an -OH group at the 21 position shown in the ABCD steroid ring system above. The numbering of carbon atoms is based on the IUPAC (International Union of Chemical Associations) Following the Union of Pure and Applied Chemistry (UNPC) accepted ring designations and atom numbering, the 21-hydroxylation of steroids is shown in Figure 1.

[0013] In a particular embodiment of the method of the first aspect of the invention, the steroid is a 3-ketosteroid. More particularly, the steroid is a non-naturally occurring steroid, i.e. a steroid that is not produced and / or 21-hydroxylated in a cell that has not been genetically modified, in particular a human or bovine cell.

[0014] In one embodiment, the steroid is selected from the group consisting of medrane, deltamedrane, progesterone, 17OH-progesterone, medroxyprogesterone, and 5-α-dihydroprogesterone. 21-hydroxylation converts these particular steroids to premedrol, medrol, 11-deoxycorticosterone, 11-deoxycortisol, 21OH-medroxyprogesterone, and 21OH-(5α-dihydroprogesterone), respectively. In a particular embodiment where the steroid is a non-naturally occurring steroid, the steroid is selected from the group consisting of medrane, deltamedrane, medroxyprogesterone, and 5-α-dihydroprogesterone.

[0015] The cells are in particular cultured cells that are cultured in any cell culture medium, such as growth medium, and in certain embodiments are quiescent. According to this embodiment, the cells are not in growth medium, but in a buffer or medium that can sustain the cells. The composition of the buffer depends on the particular cell, and suitable buffers are well known in the art. Depending on the type of cell, the cells are suspension cells or adherent cells. Suspension cells are cells that can naturally exist in suspension (i.e. without attachment to a surface) or cells that have been modified to survive in suspension culture, for example to grow to a higher density than that allowed by adherent conditions. Adherent cells are cells that require a surface, such as tissue culture plastic or microcarriers, which may be coated with extracellular matrix (such as collagen and laminin) components to improve adhesion properties and provide other signals necessary for proliferation and differentiation. In one embodiment, the adherent cells are monolayer cells.

[0016] The cells may generally be prokaryotic or eukaryotic. A particular example of a prokaryotic cell for use in the method of the first aspect is an E. coli cell, such as the E. coli strain C43(DE3) cell in the examples. A particular example of a eukaryotic cell is a yeast cell, such as an S. cerevisae cell or a Schizosaccharomyces pombe cell. However, the method of the first aspect is not limited to any particular cell type, and any cell type may be used. In particular, any cell type that can be grown and maintained in culture and that can be used as a recombinant expression system, such as insect cells or mammalian cells, in addition to the cells mentioned above, may be used.

[0017] The term "heterologous" means that a protein is expressed in a cell that does not normally (i.e., without human intervention) express that protein. For example, CYP21A2 is an enzyme also called 21-hydroxylase, which is part of the cytochrome P450 family of enzymes. Cytochrome P450 enzymes are involved in many processes in the body, such as assisting in reactions that break down drugs, as well as aiding in the production of cholesterol, certain hormones, and fats (lipids). 21-hydroxylase enzymes are found in the adrenal glands, which are located at the top of the kidney and produce various hormones that regulate many essential functions in the body. Thus, with respect to heterologous expression, CYP21A2 protein can be considered heterologous to any cell that is not an adrenal cell.

[0018] In particular, the term "heterologous" may refer to the species from which a protein or gene originates compared to the cell in which it is expressed, in particular recombinantly expressed. A heterologous protein is then a protein that originates from a different species than the cell in which it is expressed, i.e. the cell of the method of the first aspect of the invention. For example, the inventors have expressed mammalian, in particular human or bovine, proteins in E. coli cells, which makes these proteins heterologous to the cells.

[0019] In a particular embodiment of the method of the first aspect of the present invention, the CYP21A2 protein is a protein of human or bovine origin. Human CYP21A2 (UniProt accession number P08686) has a sequence according to SEQ ID NO: 1 in the sequence listing. Bovine CYP21A2 (UniProt accession number P00191) has a sequence according to SEQ ID NO: 2 in the sequence listing. See also FIG. 8. However, homologous genes are also present in other mammalian species, such as Canis lupus (dog), Macaca mulata (rhesus monkey), Rattus norvegicus (rat), Gallus gallus (chicken), Danio rerio (zebrafish), Mus musculus (mouse) or Pan troglodytes (chimpanzee). It is therefore emphasized that any CYP21A2 protein, in particular any mammalian CYP21A2 protein, can be used. Modified human CYP21A2 or bovine CYP21A2 may also be used according to SEQ ID NO: 3 and SEQ ID NO: 4, respectively. See also FIG.

[0020] The term "functional variant" refers to a protein variant that has at least 20% (e.g., at least: 20%; 30%; 40%; 50%; 60%; 70%; 80%; 90%; 95%; 98%; 99%; 99.5%; or 100%; or even more) of the ability of the native protein or CYP21A2 protein to 21-hydroxylate steroids. This ability can be determined without undue burden by those skilled in the art, for example, using the methods set forth in Examples 1 and 2 herein.

[0021] A "protein variant" is a protein having an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the CYP21A2 from which it is derived, e.g., SEQ ID NO: 1 or 3 for human CYP21A2, or SEQ ID NO: 2 or 4 for bovine CYP21A2. Determination of the percent identity of two sequences may be performed according to the method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90, 5873-5877, 1993. Such an algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215, 403-410. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402. When utilizing the BLAST and Gapped BLAST programs, the default parameters of each program are used.

[0022] Alternatively, protein variants can be defined as having no more than 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 amino acid substitutions, particularly conservative amino acid substitutions. Conservative substitution tables are well known in the art (see, for example, Creighton (1984) Proteins. WH Freeman and Company). An overview of the physical and chemical properties of amino acids is shown in Table 1 below. In certain embodiments, conservative substitutions are those made with amino acids having the same properties according to Table 1.

[0023] [Table 1]

[0024] The term "variant" also includes protein fragments. Fragments of CYP21A2 have N-terminal and / or C-terminal deletions of a total of no more than 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 amino acids. In certain embodiments, the functional variant is a fragment of CYP21A2 lacking the hydrophobic anchor region, i.e., truncated by the 29 N-terminal amino acid residues (Figure 8).

[0025] Additionally, the CYP21A2 protein may be modified, for example, by the addition of N- or C-terminal amino acids, such as a tag, or an N-terminal modification to improve bacterial expression. For example, the tag may be a C-terminal His tag, such as a 6xHis tag, and the N-terminal modification may be the addition of 10 amino acids to the N-terminus of CYP2C3 (see Figure 8 and SEQ ID NOs: 3 and 4).

[0026] An electron transport chain is a series of compounds that transfers electrons from an electron donor to an electron acceptor by a redox reaction. The term "capable of transferring electrons to CYP21A2" means that CYP21A2 is the electron acceptor of the chain. As used herein, the term "series" may include CYP21A2, and thus the chain may consist of a single protein in addition to CYP21A2. In the method of the first aspect of the invention, it is not important which members the chain consists of, so long as the chain is capable of transferring electrons to CYP21A2. Suitable systems are well known in the art. In certain embodiments of the method of the first aspect of the invention, at least one electron transport chain comprises (i) CYP21A2 reductase, or (ii) ferredoxin reductase, preferably NADPH-dependent ferredoxin reductase (adrenodoxin reductase) and ferredoxin. For example, at least one electron transport system may be a combination of NADPH-dependent cytochrome P450 reductase (CPR, e.g., human or bovine), adrenodoxin reductase (AdR, e.g., human or bovine) and adrenodoxin (Adx4-108, e.g., human or bovine), or flavodoxin reductase (Fpr, e.g., from E. coli) and adrenodoxin (Adx4-108, e.g., human or bovine). combination of an adrenodoxin reductase homolog (Arh), such as the S. pombe adrenodoxin reductase homolog (Arh1) with an adrenodoxin (Adx4-108, e.g., human or bovine); an adrenodoxin reductase homolog (Arh), such as the S. pombe adrenodoxin reductase homolog (Arh1) with the ferredoxin domain of the electron transfer domain (etp fd ), e.g., the ferredoxin domain of the S. pombe electron transfer domain (etp1 fd ), and flavodoxin reductase (Fpr, e.g., from E. coli) and the ferredoxin domain of the electron transfer domain (etp fd ), e.g., the ferredoxin domain of the S. pombe electron transfer domain (etp1 fd) and combinations thereof.

[0027] In certain embodiments, exogenous NADPH is not added to the cell. In this embodiment, NADPH is produced by the cell. In certain embodiments, NADPH production in the cell may be increased by recombinant means, such as heterologous expression of enzymes involved in NADPH production (e.g., one or more of glucose-6-phosphate dehydrogenase (G6PD), phosphogluconate dehydrogenase (PGD), malate dehydrogenase (MDH), and / or isocitrate dehydrogenase (ICDH)), and / or by providing a hormonal signal, i.e., by adding hormones such as estradiol that increase the level of endogenous NADPH production.

[0028] Similarly, the chaperone can be any chaperone as long as it is capable of promoting the folding of CYP21A2. The inventors have found that the method of the first aspect of the invention can rely only on endogenous chaperones, i.e., further expression of a suitable chaperone is not essential. However, further expression can improve the production of functional CYP21A2 in the cell and thus be beneficial. Thus, in one embodiment of the method of the first aspect of the invention, the one or more chaperones are recombinantly expressed chaperones. In particular, the one or more chaperones may be heterologous chaperones. Exemplary chaperones are the E. coli chaperones GroEL and GroES, or other chaperones such as DnaK, DnaJ, GrpE, and ClpB, and small heat shock proteins (sHSPs) such as IbpA and IbpB (IbpAB).

[0029] Optionally, the same chaperone, or one or more additional chaperones expressed in the cell, can fold one or more of the above electron transfer proteins.

[0030] Recombinant expression refers to the expression of a recombinant gene. Such a gene can be any gene that has been introduced into a cell by genetic engineering methods, and is usually a heterologous gene and / or a gene whose expression is regulated differently from the corresponding endogenous gene that may be present. In one embodiment, recombinant expression is inducible.

[0031] In one embodiment of the method of the first aspect of the invention, the method further comprises, for example after step b), adding one or more cell permeabilizing agents to the cell suspension. Cell permeabilizing agents are reagents that increase membrane permeability. Examples are organic solvents such as methanol, acetone, or DMSO, detergents such as saponin, Triton X-100, or Tween-20, and EDTA. In particular, polymyxin B can be used as a cell permeabilizing agent. This agent has been found to work particularly well in the method of the invention.

[0032] In another embodiment of the method of the first aspect of the invention, the method comprises the step of: The method further comprises a step c) of extracting the 21-hydroxylated steroids from the cell supernatant (i.e., the buffer or medium in which the cells are contained), e.g., the whole cell suspension. This extraction can be performed using any solvent or extraction method known in the art for extracting undissolved compounds. For example, solvents such as 1-butanol, 2-butanone, or chloroform may be used.

[0033] In a particular embodiment of the method of the first aspect of the invention, the expression of at least one tryptophanase gene is reduced or eliminated in the cell. Tryptophanase, i.e. L-tryptophan indole-lyase (EC number 4.1.99.1.), is an enzyme that catalyzes the reaction L-tryptophan + H2O = indole + pyruvate + NH3. Thus, reducing or eliminating expression reduces the production of indole by the cell. Indole is an inhibitor of CYP enzymes such as CYP21A2, which can improve the method. This particular embodiment is particularly suited to the embodiment of the method of the first aspect of the invention in which the cell is a prokaryotic cell, since tryptophanase genes are generally (but not exclusively) found in prokaryotes, such as E. coli. In one embodiment, the species of cells expressing the tryptophanase gene is selected from the group consisting of Aeromonas hydrophila, Bacillus sp., Bacteroides sp., Corynebacterium sp., Enterobacter aerogenes, Enterobacter aerogenes SM-18, Enterobacter sp., Erwinia sp. sp., Escherichia aurescens, Escherichia coli, Fusobacterium necrophorum subsp. Funduliforme, Kluyvera sp., Micrococcus aerogenes, Morganella morganii, Paenibacillus alvei, Paracolobactrum coliforme, Paracolobactrum sp., Pasteurella sp., Photobacterium profundum, Porphyromonas gingivalis gingivalis, Prevotella intermedia intermedia, Proteus vulgaris, Providencia rettgeri, Shigella alkalescens, Sphaerophorus sp., Symbiobacterium thermophilum, Vibrio sp., and mammalian species such as Homo sapiens and Rattus norvegicus.

[0034] In a further embodiment of the method of the first aspect of the invention, the cells further express a heterologous or recombinant gene encoding an enzyme that catalyzes a step in the biosynthetic pathway of heme, in particular the heterologous hemA (glutamyl-tRNA reductase) gene. An example of such a cell is E. coli. This advantageously reduces the need to supply precursors for CYP heme synthesis, such as the heme precursor δ-aminolevulinic acid, reducing the cost of steroid biotransformation in such cells.

[0035] In certain embodiments of the method of the first aspect of the invention, (i) a heterologous CYP21A2 TA Nucleic acids encoding a protein or functional variant thereof, (ii) at least one heterologous electron transport system capable of transferring electrons to CYP21A2, and optionally (iii) one or more chaperones that facilitate folding of CYP21A2 are included in one or more expression cassettes that are integrated into the cell, particularly the genome of the cell. The term "expression cassette" refers to a DNA fragment that includes a gene operably linked to a regulatory sequence, such as a promoter, required for gene expression. "Operably linked" refers to the linkage of nucleotide regions that code for specific genetic information such that the nucleotide regions are contiguous, the functionality of the regions is conserved, and they work in conjunction with other regions as part of a functional unit. Nucleic acids (i), (ii), and optionally (iii) may each be included in an individual expression cassette or may be included in one or more polycistronic expression cassettes. As used herein, the term "polycistronic" refers to multiple cistrons, i.e., multiple nucleic acids or genes, operably linked to the same regulatory sequence, such as a promoter.

[0036] In one embodiment, (i) a heterologous CYP21A2 protein or functional variant thereof, (ii) at least one heterologous electron transport system capable of transferring electrons to CYP21A2, and optionally (iii) one or more chaperones that facilitate folding of CYP21A2 are contained in an expression vector contained in a cell. An "expression vector" is a vehicle by which a DNA fragment containing a nucleic acid encoding a protein can be introduced into a host cell, where the host cell can express the nucleic acid. Nucleic acids (i), (ii), and optionally (iii) may each be contained in an individual expression vector, or may be contained in one or more polycistronic expression vectors.

[0037] Alternatively, one or more of nucleic acids (i), (ii), and optionally (iii) may be comprised in an expression cassette that is integrated into the genome of the cell, and the remaining nucleic acids (i), (ii), or optionally (iii) are comprised in an expression vector, both as described above.

[0038] In a second aspect, the present invention provides a cell comprising: (i) a heterologous CYP21A2 protein or a functional variant thereof; (ii) at least one heterologous electron transport system capable of transferring electrons to CYP21A2; and (iii) one or more chaperones that facilitate folding of CYP21A2 The present invention relates to a cell expressing

[0039] As with the cells of the method of the first aspect of the invention, this cell also comprises (i) a heterologous CYP21A2 protein or a functional variant thereof; (ii) at least one heterologous electron transport system capable of transferring electrons to CYP21A2; and (iii) one or more chaperones that facilitate folding of CYP21A2 One or more nucleic acids encoding The cell may be described as comprising:

[0040] In certain embodiments, these nucleic acids are comprised in one or more expression cassettes.

[0041] The cell of the second aspect is essentially a cell for use in the method of the first aspect of the invention and thus further embodiments of the cell of the second aspect of the invention are as described above in relation to the method of the first aspect.

[0042] In a third aspect, the present invention provides a polycistronic expression vector comprising: (i) a nucleic acid encoding a CYP21A2 protein or a functional variant thereof; (ii) one or more nucleic acids encoding at least one heterologous electron transport system capable of transferring electrons to CYP21A2, and optionally (iii) one or more nucleic acids encoding chaperones that facilitate folding of CYP21A2 The present invention relates to a polycistronic expression vector comprising:

[0043] In a fourth aspect, the present invention provides a kit comprising: a cell according to the second aspect, a polycistronic expression vector of the third aspect, or (i) an expression vector comprising a nucleic acid encoding a CYP21A2 protein or a functional variant thereof, (ii) one or more expression vectors comprising one or more nucleic acids encoding at least one heterologous electron transport system capable of transferring electrons to CYP21A2, and optionally (iii) one or more expression vectors comprising one or more nucleic acids encoding chaperones that facilitate folding of CYP21A2. The present invention relates to a kit comprising:

[0044] The term "kit" as used herein refers to a collection of all or part of the reagents, materials, and instructions required to carry out the method of the first aspect. The kit includes cell culture media or buffers (both dry and liquid forms), one or more cell permeabilizing agents, a solvent for steroid extraction, and in particular, one or more of hydroxylated steroids, in particular 3-ketosteroids, such as medullane, deltamedullane, progesterone, 17OH-progesterone, medroxyprogesterone, or 5α-dihydroprogesterone. Furthermore, it is contemplated that all reagents or materials described herein in connection with the method of the first aspect can be part of the kit of the fourth aspect.

[0045] In certain embodiments, the method of the first aspect, the cell of the second aspect, the expression vector of the third aspect, and the kit of the fourth aspect, if applicable, do not comprise further steps or components, in particular do not comprise heterologous genes or proteins, in particular heterologous genes or proteins for steroid conversion or steroid production not related to the 21-hydroxylation of steroids as described herein. Optionally, exceptions to this may be downstream steps or components related to further processing of 21-hydroxylated steroids (such as the conversion of premedrol to medrol), or upstream steps or components related to the production of 21-hydroxylated steroids (such as the production of medran).

[0046] In the following figures and examples, some specific embodiments of the present invention are described in more detail. However, it is not intended to limit the present invention by the details of the specific embodiments. On the contrary, the present invention relates to any embodiment including details that are not explicitly mentioned in the embodiments of this specification, but that can be found by a person skilled in the art without undue effort. [Brief description of the drawings]

[0047] [Figure 1] FIG. 1 illustrates the hydroxylation of a steroid (here, progesterone) at carbon atom 21. [Diagram 2] FIG. 1 shows a scheme of whole-cell biotransformation of steroids by CYP21A2 and required electron transfer proteins in E. coli. [Diagram 3] Left: CO difference spectrum of purified bovine CYP21. Right: SDS-PAGE of bCYP21 samples taken after the indicated purification steps (IMAC / DEAE / SP). [Figure 4] FIG. 2 shows HPLC chromatograms of the in vitro 21-hydroxylation of medran to premedrol by human CYP21 and the indicated electron transfer proteins, here AdR and Adx (system 2). [Figure 5-1] FIG. 1 shows vectors for whole-cell biotransformation constructed with different electron transfer proteins using human or bovine CYP21. [Figure 5-2] Continued from Figure 5-1. [Figure 6-1] FIG. 1 shows HPLC chromatograms of whole cell 21-hydroxylation of medran to premedrol (A), deltamedran to medrol (B), medroxyprogesterone to 21OH-medroxyprogesterone (C), and 17OH-progesterone to 11-deoxycortisol (D) by bovine CYP21 and the indicated electron transfer proteins, here Fpr and Adx. [Figure 6-2] Continued from Figure 6-1. [Figure 7] FIG. 1 shows the time dependence of whole cell conversion of medran to premedrol by bovine CYP21 and the indicated electron transfer proteins, here Fpr and Adx. [Figure 8-1] FIG. 1 shows the amino acid sequences of wild-type and modified human (A) and bovine (B) CYP21A2. [Figure 8-2] Continued from Figure 8-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] Example 1 In vitro hydroxylation 1.1 Expression / purification of hCYP21 / bCYP21 In vitro studies were performed with both human and bovine CYP21 enzymes to demonstrate that both can hydroxylate steroids at position 21. As an exemplary 21-hydroxylation method, medran was converted to premedrol. [ka]

[0049] Premedrol (methylhydrocortisone) is the precursor of the highly effective pharmaceutical steroid Medrol (methylprednisolone), an important drug in the therapy of autoimmune diseases, multiple sclerosis, and generally for the topical and systemic treatment of inflammation.

[0050] Both enzymes were expressed in E. coli strain C43(DE3) by co-expression of the E. coli chaperones GroEL / GroES encoded in vector pGro12. These chaperones ensure correct protein folding, which is important for the incorporation of the heme prosthetic group. In Figure 3, the SDS-PAGE and CO difference spectra of purified bovine CYP21 are shown. As the CO difference spectrum shows, the enzyme was purified in active form. To determine the binding of medran to both isozymes, the binding constants (K D value) was determined.

[0051] 1.2 Expression of electron delivering redox partners For efficient substrate conversion, both isoforms require a two-part electron transport chain: the cytochrome P450 enzyme itself, which is essential for the hydroxylation reaction, and one or two electron transport proteins. Without these proteins, the reaction cannot occur. For example, the six electron transport chains listed in Table 2 can transfer electrons to CYP21.

[0052] [Table 2]

[0053] All redox partners were purified for in vitro studies and validation of substrate conversion.

[0054] 1.3 Reconstitution of the cytochrome P450 system in vitro In vitro substrate conversion using purified enzymes in defined buffers and with an NADPH regenerating system has demonstrated that both isoforms, together with the electron transfer proteins listed herein, can convert medran to premedrol very efficiently. Figure 4 shows the in vitro conversion of medran by human CYP21 and electron transport system 2. The results indicate that CYP21, together with a suitable redox system, for example, as shown in Table 2, can enzymatically produce steroids exemplified by premedrol.

[0055] Example 2 Whole cell line Given the success of in vitro transformation of steroids, biotransformation in whole cells was developed.

[0056] In general, CYP21 and the necessary electron transfer proteins were heterologously expressed in E. coli strain C43(DE3) to perform the hydroxylation in whole cells. For the conversion of CYP21, bicistronic or tricistronic vectors based on the plasmid pET17b were constructed. The vectors carry the genes for a specific CYP21 and a specific redox system. Figure 5 shows all the constructed vectors. To promote correct protein folding, the E. coli chaperones GroEL and GroES were co-expressed in a second vector. The transformed E. coli cells were able to produce the CYP21 enzyme and the required redox partners. After protein expression, substrate conversion took place, which was initiated by adding a hydroxylating steroid (exemplified by medran) as a substrate.

[0057] In particular, E. coli strain C43(DE3) was transformed with vectors for whole cell biocatalysis (e.g., p21b_ArET) and pGro12 encoding the chaperones GroEL / GroES. Cultures contained 200 mL of TB medium (+ antibiotics ampicillin and kanamycin) inoculated with 2 mL of seed culture in 2 L Erlenmeyer flasks. The cultures were grown at 37°C. Expression was induced at OD0.5 by adding 1 mM IPTG, 1 mM δ-aminolevulinic acid, 4 mg / mL arabinose and maintained at 27°C for 28 h. For whole cell biotransformation, cells were harvested by centrifugation and washed with 50 mM potassium phosphate buffer (pH 7.4). Substrate conversion was initiated by adding 400 μM substrate with resting cells to 25 mL of potassium phosphate buffer (50 mM) containing 2% glycerol, 1 mM IPTG, 1 mM δ-aminolevulinic acid, and 4 mg / mL arabinose in a 300 mL baffled flask at a cell density of approximately 24 g / L (wet weight) for 24 h. Samples were taken after 24 h, and steroids were extracted with chloroform and then measured by RP-HPLC.

[0058] In Figure 6 it is shown that the steroids were converted to their 21-hydroxylated derivatives and, in contrast to chemical synthesis, no appearance of by-products was observed. The time dependence of product formation was studied for each CYP21 isoform in whole cells with six redox systems to determine not only the end-point yields but also the reaction kinetics, which are of great interest for biotechnological methods (Figure 7).

[0059] Following the conversion of medran to premedrol, both human and bovine CYP21 were able to hydroxylate all 3-ketosteroids tested that were not already hydroxylated at position 21. In particular, the following steroid conversions could be demonstrated: Medran to Premedrol (non-natural substrate) Deltamedran to Medrol (non-natural substrate) Progesterone to 11-deoxycorticosterone (natural substrate) 17OH-progesterone to 11-deoxycortisol (natural substrate) Medroxyprogesterone to 21OH-medroxyprogesterone (unnatural substrate) 5α-dihydroprogesterone to 21OH-(5α-dihydroprogesterone)

Claims

1. 1. A method for hydroxylating carbon atom 21 of a steroid, comprising the steps of: a) (i) a heterologous mammalian CYP21A2 protein or a functional variant thereof having at least 20% of the ability to 21-hydroxylate steroids of an unmodified heterologous mammalian CYP21A2 protein; (ii) at least one heterologous electron transport system capable of transferring electrons to CYP21A2, - a combination of flavodoxin reductase (Fpr) and adrenodoxin, - Fpr and the ferredoxin domain of the electron transfer domain (etp fd ) in combination with - combination of adrenodoxin reductase homologue (Arh) with adrenodoxin, and - Arh and etp fd In combination with A heterologous electron transport system selected from the group consisting of: (iii) providing a prokaryotic cell expressing one or more chaperones that facilitate folding of CYP21A2; and b) adding a steroid to the prokaryotic cell; The method comprising:

2. The method of claim 1, further comprising step c) of extracting 21-hydroxylated steroids from the supernatant of the prokaryotic cells.

3. 3. The method of claim 1 or 2, further comprising adding one or more cell permeabilization agents to the prokaryotic cells after step b).

4. The method according to any one of claims 1 to 3, wherein the steroid is a 3-keto steroid.

5. 5. The method of claim 4, wherein the 3-ketosteroid is selected from the group consisting of medullane, deltamedullan, progesterone, 17OH-progesterone, medroxyprogesterone, and 5-α-dihydroprogesterone.

6. A method according to any one of claims 1 to 5, wherein the prokaryotic cell is a resting cell.

7. The method according to any one of claims 1 to 6, wherein the prokaryotic cell is an Escherichia coli cell.

8. - Fpr is E. coli Fpr, - the adrenodoxin is human or bovine Adx4-108, - Arh is S. pombe adrenodoxin reductase homologue (Arh1), and / or - etp fd The ferredoxin domain of the S. pombe electron transfer domain (etp1 fd ) The method according to any one of claims 1 to 7.

9. The method of any one of claims 1 to 8, wherein the one or more chaperones are recombinantly expressed chaperones.

10. The method of any one of claims 1 to 9, wherein expression of at least one tryptophanase gene is reduced or eliminated in the prokaryotic cell.

11. The method according to any one of claims 1 to 10, wherein the prokaryotic cell further expresses a heterologous gene, in particular the hemA gene, encoding an enzyme that catalyzes a step in the heme biosynthetic pathway.

12. 12. The method of any one of claims 1 to 11, wherein the genes encoding (i), (ii) and optionally (iii) are contained in one or more expression cassettes that are integrated into the genome of the cell.

13. A prokaryotic cell comprising: (i) a heterologous mammalian CYP21A2 protein or a functional variant thereof having at least 20% of the ability of an unmodified heterologous mammalian CYP21A2 protein to 21-hydroxylate steroids; (ii) at least one heterologous electron transport system capable of transferring electrons to CYP21A2, - a combination of flavodoxin reductase (Fpr) and adrenodoxin, - Fpr and the ferredoxin domain of the electron transfer domain (etp fd ) in combination with - combination of adrenodoxin reductase homologue (Arh) with adrenodoxin, and - Arh and etp fd In combination with A heterologous electron transport system selected from the group consisting of: (iii) one or more chaperones that facilitate folding of CYP21A2. The prokaryotic cell expressing 14. A kit comprising the prokaryotic cell of claim 13.

15. 15. The kit of claim 14, comprising one or more nucleic acids encoding chaperones that facilitate folding of CYP21A2.

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