Biotechnological optimization of microorganisms for 1,2-dehydrogenation of steroids

Abstract

The present invention relates to genetically modified bacteria and their industrial applications, particularly in the 1,2-dehydrogenation of steroids.

Description

[Technical field]

[0001] The present invention relates to genetically modified bacteria and their industrial use, particularly in the 1,2-dehydrogenation of steroids. [Background technology]

[0002] The 1,2-dehydrogenation of steroids (also called Δ1-dehydrogenation) can be carried out by biotransformation using bacteria with the appropriate enzyme system. For example, this transformation can be catalyzed by microorganisms of the Nocardioidaceae family, which are produced and used as bacterial suspensions or as supernatants after cell lysis of bacterial suspensions. Unfortunately, microorganisms also express enzymes that catalyze a second (undesired) reaction on the steroid molecule, which is the reduction of the keto group at the 20-position, resulting in the production of a by-product referred to herein as the "20-OH impurity". These two reactions are diagrammed hereinafter using the example of hydrocortisone. [ka]

[0003] The enzyme 20β-HSDH (20-beta-hydroxysteroid dehydrogenase), which effects ketoreduction at the 20-position in steroids, has been identified as responsible for this side reaction. Summary of the Invention [Problem to be solved by the invention]

[0004] The present inventors have identified the gene responsible for the side reaction activity and genetically engineered a microorganism that does not have this activity. When used in steroid conversion, it no longer produces unwanted by-products, and the genetically engineered microorganism according to the present invention described above can be used directly in steroid conversion without any potential maturation step. Further steps for purification of the product to remove unwanted by-products can also be omitted. The product of the biotransformation can be used as it is after production. In conclusion, this leads to higher product yields, higher product quality, and a simpler production process. [Means for solving the problem]

[0005] Description of the invention Thus, a first aspect of the present invention relates to a genetically modified bacterium in which expression of a gene encoding 20β-HSDH is reduced or suppressed compared to a corresponding unmodified bacterium, wherein the corresponding unmodified bacterium has Δ1-dehydrogenase activity and 20-β-dehydrogenase activity.

[0006] As used herein, the term " Corresponding unmodified bacteria " indicates the same bacterial strain as the unmodified bacteria that is subsequently subjected to genetic modification.

[0007] term" bacteria" refers to a prokaryotic microorganism. The unmodified bacterium has 20β-HSDH activity and Δ1-dehydrogenase activity. In one embodiment, the bacterium is from an order selected from among the Actinomycetales order, Clostridiales order, Aeromonadales order, Pasteurellales order, Bacillales order, Lactobacillales order, Bifidobacteriales order and Coriobacteriales order. In one preferred embodiment, the bacterium is from the Actinomycetales order. For example, the bacterium may be from the Nocardiodiaceae family, such as Nocardioides simplex, the Streptomycetaceae family, such as Streptomyces albus, or the Mycobacteriaceae family, such as Mycobacterium avium, and in particular Mycobacterium avium subsp. paratuberculosis MAP4. In a particular embodiment, the bacterium is from the Nocardioides family, preferably the genus Nocardioides, and most preferably the bacterium is Nocardioides simplex.

[0008] term" Bacteria from the Nocardioides family" means any bacterium from this family having 20β-HSDH activity and Δ1-dehydrogenase activity, for example a bacterium from the genera Actinopolymorpha, Aeromicrobium, Flindersiella, Friedmanniella, Kribbella, Marmoricola, Micropruina, Mumia, Nocardioides, Pimelobacter, Propionicicella, Propionicimonas, Tenggerimyces or Thermasporomyces. Preferably, the bacterium from the Nocardioides family is Nocardioides simplex (previously called Arthrobacter simplex or Corynebacterium simplex or Pimelobacter simplex). Preferably, the bacterium is Nocardioides simplex ATCC6946. Other preferred bacteria include Friedmanniella flava (Taxonomy ID: 1036181), Friedmanniella sagamiharensis (Taxonomy ID: 546874), Nocardioides dokdonensis FR1436 (Taxonomy ID: 1300347), Nocardioides lianchengensis (Taxonomy ID: 1045774), Nocardioides psychrotolerans (Taxonomy ID: 1005945), Nocardioides szechwanensis (Taxonomy ID: 1006666), Nocardioides szechwanensis (Taxonomy ID: 1005945 ...ID:1005944), Corynebacterium coyleae (Taxonomy ID:53374), Corynebacterium glyciniphilum AJ 3170 (Taxonomy ID:1404245), Corynebacterium kroppenstedtii DSM 44385 (Taxonomy ID:645127), Corynebacterium lowii (Taxonomy ID:1544413), Corynebacterium spheniscorum (Taxonomy ID:1544413), ID:185761), Corynebacterium vitaeruminis DSM 20294 (Taxonomy ID:1224164), Aeromicrobium marinum DSM 15272 (Taxonomy ID:585531), Nocardioides luteus (Taxonomy ID:1844), Corynebacterium halotolerans YIM 70093 = DSM 44683 (Taxonomy ID:1121362), Corynebacterium maris DSM 45190 (Taxonomy ID:1121362) ID: 1224163) and Corynebacterium riegelii (Taxonomy ID: 156976).

[0009] term" gene" means a DNA sequence that codes for or corresponds to a specific sequence of amino acids comprising all or part of one or more proteins or enzymes, and which may or may not include regulatory DNA sequences, such as promoter sequences, which determine the conditions under which the gene is expressed. In particular, the term gene may be intended to mean a genomic sequence that codes for a protein, i.e., a sequence including regulatory elements, promoter, intron and exon sequences.

[0010] As used herein, the term " The gene encoding 20β-HSDH " corresponds to a gene encoding 20β-HSDH of any bacteria. Preferably, the gene encoding 20β-HSDH has at least 60% identity with the sequence of SEQ ID NO: 1, more preferably, greater than 70%, 75%, 80%, 85%, 90%, 95%, and most preferably, greater than 99% identity with the sequence of SEQ ID NO: 1. According to one embodiment, the gene encoding 20β-HSDH has at least 60% identity with the sequence of SEQ ID NO: 3, more preferably, greater than 70%, 75%, 80%, 85%, 90%, 95%, and most preferably, greater than 99% identity with the sequence of SEQ ID NO: 3.

[0011] " Genes with at least x% identity to the reference sequence By ", it is intended that the nucleic acid sequence differs from the reference sequence by up to 100-x nucleotide changes for every 100 nucleotides of the reference sequence over its entire length. In other words, up to 100-x% of the nucleotides in the subject sequence may be inserted, deleted or substituted with another amino acid or nucleotide to obtain a nucleic acid having at least x% sequence identity with the reference sequence.

[0012] A nucleic acid sequence that is "at least 60%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical" to a reference sequence may contain mutations such as deletions, insertions and / or substitutions compared to the reference sequence. In case of substitutions, the substitutions are preferably silent or correspond to substitutions that result in conservative substitutions in the translated amino acid sequence.

[0013] Methods for comparing the identity of two or more sequences are well known in the scientific community. For example, programs available in the Wisconsin Sequence Analysis Package, version 9.1, such as the programs BESTFIT and GAP, can be used to determine the identity percentage between two sequences. BESTFIT uses the "local homology" algorithm of Smith and Waterman and finds the best single region of similarity between two sequences. Other programs for determining identity between sequences are also known in the art, such as the Needle program, which is based on the algorithm of Needleman and Wunsch described in Needleman and Wunsch (1970) J. Mol Biol. 48:443-453, and uses, for example, the following parameters for polynucleotide sequence comparison: comparison matrix: DNAFULL; gap open penalty=10, gap extension penalty=0.5, end gap penalty: false, end gap open penalty=10, end gap extension penalty=0.5.

[0014] term" Genetically modified " means any detectable change in genetic material, e.g., DNA, RNA, cDNA, or any process, mechanism, or the result of such a change. It includes genetic mutations in which the structure of a gene (e.g., DNA sequence) is altered, any gene or DNA resulting from any mutational process, and any expression product (e.g., protein or enzyme) expressed by an altered gene or DNA sequence. Generally, mutations are identified in a subject by comparing the sequence of a nucleic acid or polypeptide expressed by the subject with the corresponding nucleic acid or polypeptide in a control population.

[0015] term" The expression of the gene encoding 20β-HSDH is reduced or suppressed." refers to a reduction or inhibition of the expression levels of messenger RNA (mRNA) and / or protein encoded by the gene encoding 20β-HSDH. Expression of the gene encoding 20β-HSDH can be reduced by at least 75%, 80%, 85%, 90% or 95% compared to the unmodified bacterium. Preferably, expression of the gene encoding 20β-HSDH is reduced by 100%, thus corresponding to inhibition of expression.

[0016] The expression level of a gene or nucleic acid can be measured by methods well known to those of skill in the art, including direct hybridization-based assays and amplification-based assays, among others.

[0017] The expression of the gene encoding 20β-HSDH can be reduced or suppressed compared to the unmodified bacterium by inactivation or deletion of said gene. In a particular embodiment, the gene encoding 20β-HSDH is mutated, and preferably truncated.

[0018] Preferably, the genetically modified bacteria does not contain any foreign DNA.

[0019] Laboratory procedures for gene inactivation, deletion or truncation are well known in the art.

[0020] As contemplated herein, when a gene encoding 20β-HSDH is mutated and / or truncated, it may not encode a fully functional 20β-HSDH protein, but may encode a 20β-HSDH having lower activity than the corresponding native 20β-HSDH protein when measured under the same conditions, and in particular the encoded 20β-HSDH protein may have no activity.

[0021] The genetically modified bacteria according to the invention preferably have Δ1-dehydrogenase activity but have reduced or no 20-β-dehydrogenase activity compared to unmodified bacteria.

[0022] Thus, as will be readily apparent in the present application, the present invention relates to a genetically modified bacterium, wherein: the corresponding unmodified bacterium has Δ1-dehydrogenase activity and 20-β-dehydrogenase activity, - at least one genetic modification induces a reduced or suppressed expression of a gene encoding 20-beta-hydroxysteroid dehydrogenase (20β-HSDH); and - preferably, said genetically modified bacterium maintains its Δ1-dehydrogenase activity.

[0023] Preferably, the genetically modified bacteria according to the present invention have Δ1-dehydrogenase activity but, due to the genetic modification, have reduced 20-β-dehydrogenase activity or no 20-β-dehydrogenase activity compared to unmodified bacteria.

[0024] A second aspect of the present invention relates to a method for producing a genetically modified bacterium according to the present invention, said method comprising: a) providing a bacterium having Δ1-dehydrogenase activity and 20-β-dehydrogenase activity; and b) Inactivating or deleting the gene encoding 20β-HSDH Includes.

[0025] Step b of the method for producing a genetically modified bacterium according to the invention may consist in the mutation, truncation or deletion of the gene encoding 20β-HSDH. The mutation or truncation may be achieved by any method known to the person skilled in the art, and preferably by double crossover of the gene encoding 20β-HSDH.

[0026] Preferably, the resulting genetically modified bacterium does not contain any foreign DNA.

[0027] In another aspect, the present invention provides a method for producing a composition comprising: a) providing steroids that are not 1,2-dehydrogenated; b) contacting said non-1,2-dehydrogenated steroid with a bacterium according to the invention or an extract thereof under conditions sufficient to obtain a 1,2-dehydrogenated steroid. The present invention relates to a method for producing a 1,2-dehydrogenated steroid, comprising the steps of:

[0028] Preferably, the non-1,2-dehydrogenated steroid used in the process for producing a 1,2-dehydrogenated steroid according to the present invention is a corticosteroid.

[0029] In certain embodiments, the 1,2-dehydrogenated steroid used in the method is progesterone, testosterone, androstenedione, or derivatives of these molecules that contain a steroid backbone that contains a 6 carbon atom ring A that is not dehydrogenated between the 1,2 positions.

[0030] In certain embodiments, the non-1,2-dehydrogenated steroid is: - 11beta 17,21-trihydroxypregn-4-ene-3,20-dione (hydrocortisone) and said process is for the preparation of 11beta,17alpha,21-trihydroxy-1,4-pregnadiene-3,20-dione (prednisolone); or - 17alpha,21-dihydroxy-4-pregnene-3,11,20-trione (cortisone) and said process is for the manufacture of 17alpha,21-dihydroxy-1,4 pregnadiene-3,11,20-trione (prednisone); or pregna-4-ene-3,20-dione and the process is for the preparation of pregna-1,4-diene-3,20-dione; or - 11beta,17alpha,21-trihydroxy-6alpha-methyl-1,4-pregnene-3,20-dione (6-methyl-hydrocortisone) for the manufacture of 11beta,17alpha,21-trihydroxy-6alpha-methyl-1,4-pregnadiene-3,20-dione (6-methylprednisolone); or - 9,11-dehydrocortexolone and said process is for the preparation of 1,2-9,11-dehydrocortexolone; or - 9,11 dehydrocortexolone-17,21-diacetate and said process is for the preparation of 1,2-9,11 dehydrocortexolone; or - 9,11-16,17-dehydrocortexolone-21-acetate and said process is for the preparation of 1,2-9,11-16,17-dehydrocortexolone-21-acetate; or - 16-alpha-methylhydrocortisone, and the above process is for the preparation of 16-alpha-methylprednisolone.

[0031] In one embodiment, step b) of the process for the preparation of 1,2-dehydrogenated steroids is carried out using an extract of a bacterium according to the invention, which extract has preferentially Δ1-dehydrogenase activity and preferentially reduced or no 20-β-dehydrogenase activity. In particular, such an extract can be obtained by cell lysis of a bacterium according to the invention and, optionally, fractionating the cell lysis product in order to enrich the Δ1-dehydrogenase activity or to facilitate the purification of the Δ1-dehydrogenase activity.

[0032] The process for producing a 1,2-dehydrogenated steroid may further comprise recovering and purifying the 1,2-dehydrogenated steroid.

[0033] Preferably, the process for producing 1,2-dehydrogenated steroids does not include the step of removal of 20β-dehydrogenated steroids. Indeed, this step may be unnecessary if the expression of the gene encoding 20β-HSDH is significantly reduced.

[0034] Additionally, the method for producing a 1,2-dehydrogenated steroid may include formulating the 1,2-dehydrogenated steroid into a pharmaceutical composition.

[0035] In another aspect, the present invention relates to the use of the bacterium according to the invention or an extract thereof for producing a 1,2-dehydrogenated steroid from a non-1,2-dehydrogenated steroid.

[0036] The extract has preferentially Δ1-dehydrogenase activity and preferentially reduced or no 20-β-dehydrogenase activity. In particular, such an extract can be obtained by lysis of the bacterial cells according to the invention and, optionally, after fractionation of the cell lysate in order to enrich for or facilitate the purification of the Δ1-dehydrogenase activity.

[0037] Preferably, the non-1,2-dehydrogenated steroid is a corticosteroid.

[0038] In certain embodiments, the steroid that has not been 1,2-dehydrogenated is progesterone, testosterone, androstenedione, or derivatives of these molecules that contain a steroid backbone that contains a 6 carbon atom ring A that has not been dehydrogenated between the 1,2 positions.

[0039] In certain embodiments, the non-1,2-dehydrogenated steroid is: - 11beta 17,21-trihydroxypregn-4-ene-3,20-dione (hydrocortisone) and 11beta,17alpha,21-trihydroxy-1,4-pregnadiene-3,20-dione (prednisolone) is produced; or - 17alpha,21-dihydroxy-4-pregnene-3,11,20-trione (cortisone) and 17alpha,21-dihydroxy-1,4 pregnadien-3,11,20-trione (prednisone) is produced; or - pregn-4-ene-3,20-dione and pregna-1,4-diene-3,20-dione is produced; or - 11beta,17,21-trihydroxy-6alpha-1,4-pregnene-3,20-dione (6-methyl-hydrocortisone) and 11beta,17alpha,21-trihydroxy-6alpha-methyl-1,4-pregnadiene-3,20-dione (6-methylprednisolone) is produced; or - 9,11-dehydrocortexolone and 1,2-9,11-dehydrocortexolone is produced; or - 9,11 dehydrocortexolone-17,21-diacetate, and 1,2-9,11 dehydrocortexolone is produced; or - 9,11-16,17-dehydrocortexolone-21-acetate and 1,2-9,11-16,17-dehydrocortexolone-21-acetate is produced; or - 16-alpha-methylhydrocortisone, and 16-alpha-methylprednisolone are produced.

[0040] As used herein, the term " Pharmaceutical Compositions " refers to the preparation of 1,2-dehydrogenated steroids with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of said 1,2-dehydrogenated steroids to an organism.

[0041] The pharmaceutical compositions of the present invention may be manufactured by methods that are well known in the art, for example, by means of conventional mixing, dissolving, granulating, comminuted, pulverizing, dragee-making, elutriating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0042] Typically, a pharmaceutical composition will include a 1,2-dehydrogenated steroid of the invention and a pharma- ceutically acceptable vehicle.

[0043] The invention is further illustrated by the following figures and examples. [Brief description of the drawings]

[0044] [Figure 1] Figure 1: Schematic representation of gene inactivation by double crossover. A plasmid containing a truncated form of the gene of interest (arrow containing the letter "T") flanked by two homologous shoulders (box containing a box) and the apramycin resistance gene and β-glucuronidase (arrow containing a vertical line and a wavy line) is brought into contact with the bacterial chromosome, and one of the homologous shoulders recombines with the respective sequence in the chromosome (step 1). This step results in the formation of an intermediate single crossover construct, and the entire plasmid is integrated into the genome (enclosed by a dotted line). This strand then contains two mutants of the gene of interest and is resistant to apramycin and β-glucuronidase activity. After several incubations (step 2), the two remaining homologous shoulders interact with each other (dotted lines) and a double crossover occurs (step 3). The resulting strand then contains only the truncated form of the gene (arrow containing the letter "T"). [Diagram 2] Figure 2: HPLC chromatogram of the product obtained after the classical process for producing prednisolone from hydrocortisone using Nocardioides simplex (N. simplex). The arrow indicates the 20-OH prednisolone impurity. [Diagram 3]Figure 3: HPLC chromatogram of the product obtained after the classical process for the production of prednisolone from hydrocortisone using the double-crossover mutant NOSIM5169 of N. simplex. The arrow points to the position where the 20-OH prednisolone impurity is usually observed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] Sequence Listing: SEQ ID NO:1 corresponds to the sequence of NOSIM5169 of N. simplex. SEQ ID NO:2 corresponds to the sequence of NOSIM3735 of N. simplex. SEQ ID NO: 3 corresponds to the sequence of the 20β-HSDH gene of Mycobacterium avium subsp. paratuberculosis MAP4. EXAMPLES

[0046] By analyzing the genome of N. simplex, two possible genes for 20β-HSDH were found, named NOSIM5169 (corresponding to the sequence of SEQ ID NO: 1) and NOSIM3735 (corresponding to the sequence of SEQ ID NO: 2), both of which share similarity with the sequence of the gene responsible for 20β-HSDH of M. avium subsp. paratuberculosis MAP4 (corresponding to the sequence of SEQ ID NO: 3).

[0047] 1. Plasmid construction (NOSIM5169) A plasmid for inactivation of NOSIM5169 was constructed as follows: homologous shoulders of 2.0 and 1.7 kbp in size were amplified by PCR. The primers used for amplification of one shoulder contained XbaI and EcoRV restriction sites, and the primers for the second homologous shoulder were designed to contain EcoRV and EcoRI restriction sites.

[0048] The shoulder was then cloned into the first plasmid.

[0049] To obtain a new second plasmid, the gene for hygromycin resistance was blunt-end cloned into the EcoRV restriction site.

[0050] The plasmid not containing the hygromycin resistance gene was designated "second plasmid-hyg."

[0051] 2. Electroporation Protocol for N. simplex This protocol is based on the procedure described for Arthrobacter (Zhang et al., 2011). The optimized procedure for the production of electrocompetent cells was as follows: Cells of N. simplex were inoculated into 20 ml of LB+Ph (LB stands for lysogeny broth and Ph stands for fosfomycin) and cultured until stationary phase was reached. Then 1% inoculum from the preculture was inoculated into fresh LB medium and cultured until an optical density of 0.4-0.5 at 600 nm was reached. Ampicillin (Ap) and glycine were then added to a final concentration of 30 μg / I and 5 g / I, respectively, and the culture was continued. After 3 h, the culture was placed on ice and harvested by centrifugation (4500 rpm, 6 min, 4 °C). After washing three times in ice-cold electroporation buffer (0.5 M sorbitol, 10% glycerol), the cells were concentrated 100-fold by centrifugation. Finally, the suspension was distributed into 60 μl aliquots and stored on ice (this protocol was used for the generation of N. simplex NOSIM5169-hyg).

[0052] Alternatively, the protocol for Streptomyces albus ATCC 21838 (Izumikawa et al., 2003) can be applied. In this case, the procedure for the preparation of electrocompetent cells was modified to fit N. simplex physiology: 1% inoculum from a stationary-phase preculture of N. simplex was inoculated into fresh LB+Ph medium and cultivated. When the optical density reached 0.4–0.5 at 600 nm, the culture was harvested by centrifugation (4500 rpm, 6 min, 4 °C) and washed three times with sterile ice-cold water. The mycelium was then suspended in 25 ml of cold electroporation buffer (10% sucrose, 15% glycerol) and lysozyme (0.5 mg / ml) was added. After 20 min of incubation at 37 °C, the cells were collected by centrifugation and concentrated 100-fold in electroporation buffer. Finally, the suspension was distributed into 60 μl aliquots and stored on ice.

[0053] The optimized electroporation procedure in both cases was as follows: Electrocompetent cells were mixed with 500 ng of DNA (prepared from E. coli GB2005) and the mixture was transferred to a pre-cooled electroporation cuvette (0.1 cm electrode). The mixture was subjected to a single pulse at 2.1 kV / cm using an Eppendorf pulser. Immediately after the electric pulse, the cell suspension was transferred into an Eppendorf tube containing 800 μl of recovery medium (SOC) at room temperature and incubated at 30° C. for 12 h with shaking at 850 rpm. For selection of transformed cells, serial dilutions of bacteria were spread on LB agar plates containing apramycin (Am). In each set of experiments, a negative control was performed by omitting the addition of plasmid DNA.

[0054] 3. Single crossover mutant (NOSIM5169) After electroporation of the N. simplex strain with the second plasmid, single crossover transformants were obtained and applied to LB+Am+X-Gluc agar plates (see FIG. 1 for a schematic diagram) (X-Gluc stands for monohydrate, X-GlcA). The ability to grow on apramycin and to perform the β-glucuronidase (GUS) (e.g., Myronovskyi et al., 2011) reaction confirmed that the transformants were single crossover mutants. After this, the mutants were plated in 100 ml of LB+Ph medium and incubated for 48 hours at 28° C. and 200 rpm.

[0055] 4. Double crossover mutant (NOSIM5169) Then, 1 ml of the culture was transferred into fresh LB+Ph medium and incubated under the same conditions for another 48 hours. This process was repeated several times. After the final incubation, serial dilutions of the mutants were made on LB+X-Gluc agar plates.

[0056] The mutants that did not show GUS activity were transferred to fresh LB+X-Gluc and checked again. The absence of GUS activity means that double crossover had occurred in those mutants and they had lost the plasmid backbone.

[0057] Mutants that passed the second test were grown in LB, and their gDNA was isolated and then analyzed by PCR using primers homologous to the chromosomal DNA of N. simplex.

[0058] 5. A double-crossover mutant (NOSIM5169) that does not contain foreign DNA Instead of the second plasmid, the transformation was performed using "second plasmid-hyg". The presence of an antibiotic resistance gene (hygromycin resistance) may facilitate further identification of double-crossover mutants. However, this resistance gene may be inactive in the genome of N. simplex.

[0059] Therefore, after double crossover, the resulting mutants should not contain any foreign DNA and would be available for further industrial applications (Figure 1).

[0060] Apramycin-resistant colonies were obtained after introduction of the "second plasmid-hyg" into N. simplex.

[0061] This mutant was passaged through six cultures in LB+Ph medium, followed by serial dilutions of the cultures in LB+X-Gluc.

[0062] The resulting colonies that were unable to grow on apramycin and had no GUS activity were subjected to further analysis by PCR. Amplification was performed using two primers homologous to regions of N. simplex gDNA flanking NOSIM5169.

[0063] 6. Inactivation of NOSIM3735 The previously described method for the construction of a strain in which NOSIM3735 was inactivated could not be applied because the DNA region upstream of NOSIM3735 was not accessible for its amplification by PCR. To overcome this challenge, a cosmid library containing fragments of the N. simplex genome was ordered and sequenced.

[0064] To inactivate NOSIM3735, a gene containing appropriate cosmid from a cosmid library was identified (Sanger sequencing of about 1500 cosmids by GATC Biotech, Konstanz) and the gene was replaced with the chloramphenicol (Cm) resistance gene cat using lambda-mediated recombination in E. coli (e.g., Murphy, 1998). The modified cosmid was then digested with BamHI endonuclease. In this way, a mixture of fragments was obtained, among which a fragment containing the cat gene instead of NOSIM3735 flanked by two 3 kb shoulders for homologous recombination with the N. simplex genome was obtained. The fragments were separated by agarose gel electrophoresis, and the 7-8 kb fraction was purified and ligated into a BamHI-linearized plasmid. To select mutants containing the construct containing the cat gene, LB+Am+Cm was used as the growth medium. The obtained mutants were analyzed by PCR and sequenced. One of the resulting plasmids was further used for the inactivation of NOSIM3735.

[0065] The inactivation was carried out according to the protocol previously adapted for the inactivation of NOSIM 5169 (see point 2 and below).

[0066] This mutant was passaged through several cultures in LB+Ph medium, followed by serial dilutions of the cultures in LB+X-Gluc.

[0067] The resulting colonies, which were unable to grow on apramycin and had no GUS activity, were subjected to further analysis by PCR. Amplification was performed by using two primers homologous to regions of N. simplex gDNA flanking NOSIM3735.

[0068] 7. Use of NOSIM 3735 and NOSIM 5169 for hydrocortisone The clone is used in a classical process for the production of prednisolone from hydrocortisone.

[0069] Briefly, hydrocortisone was mixed with a solution of menadione in tetrahydrofuran. This suspension was then mixed in a phosphate buffer adjusted to pH 8.0. The bacterial suspension was then added. After incubation at 30°C under stirring, the reaction was stopped by adding phosphoric acid. The increase in prednisolone was measured and analyzed by HPLC.

[0070] HPLC chromatograms were obtained on a Waters-Xbridge RP18 column using a water / acetonitrile gradient as the mobile phase (chromatograms are shown in Figures 2-3).

[0071] Alternatively, an activity assay for the production of the 20-OH-byproduct was realized. Prednisolone was suspended in phosphate buffer at pH 8.0 and incubated with the bacterial suspension. The reaction was stopped after 60 min by adding phosphoric acid. The potential increase in the 20-OH keto-reduction product was then measured by HPLC.

[0072] Truncation of NOSIM5169 globally inhibits 20β-HSDH activity and thus makes it possible to obtain prednisolone free of the 20-OH impurity (Figure 3).

[0073] The NOSIM3735 gene was knocked out, but this strain showed the same reaction as the wild-type strain, and knocking out this gene prevented unnecessary side reactions. I couldn't .

[0074] Reference list: Izumikawa, M.; Murata, M.; Tachibana, K.; Ebizuka, Y. and Fujii, I. (2003): Cloning of Modular Type I Polyketide Synthase Genes from Salinomycin Producing Strain of Streptomyces albus.Bioorganic & Medicinal Chemistry 11:3401-3405. Murphy、K.C.(1998):Use of bacteriophage λ recombination functions to promote gene replacement in Escherichia coli.J.Bacteriol.180:2063-2071. Myronovskyi、M.;Welle、E.;Fedorenko、V.and Luzhetskyy、A.(2011):Glucuronidase as a Sensitive and Versatile Reporter in Actinomycetes.Applied and Environmental Microbiology、Aug.2011、5370-5383. Zhang、H.;Li、Y.;Chen、X.;Sheng、H.and An、L.(2011):Optimization of electroporation conditions for Arthrobacter with plasmid PART2、Journal of Microbiological Methods、84:114-120.

Claims

1. A genetically modified bacterium from the Nocardioidea family that has been genetically modified to have no 20-beta-hydroxysteroid dehydrogenase (20β-HSDH) activity or reduced 20β-HSDH activity compared to a corresponding unmodified bacterium having Δ1-dehydrogenase activity and 20-β-dehydrogenase activity, wherein the corresponding unmodified bacterium contains a gene encoding a 20β-HSDH having at least 90% identity to the sequence of SEQ ID NO:1, and wherein the genetic modification inactivates or deletes the gene encoding a 20β-HSDH having at least 90% identity to the sequence of SEQ ID NO:

1.

2. The genetically modified bacterium of claim 1 , wherein the gene encoding 20β-HSDH is mutated.

3. 3. The genetically modified bacterium according to claim 1 or 2, wherein the gene encoding 20β-HSDH is truncated.

4. The genetically modified bacterium according to any one of claims 1 to 3, wherein expression of a gene encoding 20β-HSDH is reduced or suppressed.

5. The genetically modified bacterium according to any one of claims 1 to 4, wherein the genetically modified bacterium has Δ1-dehydrogenase activity.

6. a) providing a bacterium from the Nocardioides family having Δ1-dehydrogenase activity and 20-β-dehydrogenase activity; and b) inactivating or deleting a gene encoding a 20β-HSDH having at least 90% identity with the sequence of SEQ ID NO:1; A method for producing the genetically modified bacterium according to any one of claims 1 to 5, comprising:

7. Step b) is a step of determining whether a mutation, truncation or deletion of the gene encoding 20β-HSDH has occurred. The method of claim 6 , wherein the method is realized by:

8. a) providing a steroid that is not 1,2-dehydrogenated; b) contacting said non-1,2-dehydrogenated steroid with a bacterium or an extract thereof according to any one of claims 1 to 5 under conditions sufficient to obtain a 1,2-dehydrogenated steroid. A method for producing a 1,2-dehydrogenated steroid, comprising:

9. The method of claim 8, wherein the non-1,2-dehydrogenated steroid is a corticosteroid.

10. 10. The process according to claim 8 or 9, wherein the non-1,2-dehydrogenated steroid is hydrocortisone and the process is for the preparation of prednisolone.

11. 11. The method of any one of claims 8 to 10, further comprising recovering and purifying the 1,2-dehydrogenated steroid.

12. 12. The method of claim 11, which does not include a step of removing 20β-dehydrogenated steroids.

13. 13. The method of any one of claims 8 to 12, further comprising formulating the 1,2-dehydrogenated steroid into a pharmaceutical composition.

14. The method according to any one of claims 8 to 13, wherein the extract has Δ1-dehydrogenase activity.

15. 15. The method according to any one of claims 8 to 14, wherein the extract is obtained by cell lysis of the bacterium according to any one of claims 1 to 5 and optionally fractionating the cell lysis product in order to enrich for Δ1-dehydrogenase activity or to facilitate the purification of the Δ1-dehydrogenase activity.

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