RU2677332C1 - Recombinant strain of filamentous fungus aspergillus nidulans and its use for progesterone 11α-hydroxylation and for acetylation of 11α-hydroxyprogesterone - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology and biochemistry, namely to recombinant mycelial fungus Aspergillus nidulans 031/pDHG25-SgrDI (pyrG) strain. This strain has steroid-11α-hydroxylating activity, the ability to convert progesterone into 11α-acetoxyprogesterone, acetylating activity and ability to esterify 11α-hydroxyprogesterone. Present strain was obtained by transforming strain A. nidulans 031 (argB; pyrG) (AN031;CBS 129193) autonomously replicable plasmid pDHG25-SgrDI containing a unique SgrDI restriction site. Invention also relates to use of said strain for 11α-hydroxylation of progesterone to form 11α-hydroxyprogesterone and for acetylation 11α-hydroxyprogesterone to give 11α-acetoxyprogesterone.EFFECT: present invention allows to obtain 11α-hydroxyprogesterone and 11α-acetoxyprogesterone with the help of biocatalytic processes.3 cl, 23 dwg, 1 tbl, 6 ex

Description

Technical field

The present invention relates to the field of biotechnology, as well as to genetics and molecular biology, specifically to cells of living cultures, including recombinant ones, and relates to the creation and use of a new recombinant strain of mycelial fungus for hydroxylation and acetylation of steroid compounds, in particular, compounds of the pregnan series (progesterone derivatives ), and can be used in industrial biotechnology, as well as in the pharmaceutical industry, for the production of steroid medications.

State of the art

The introduction of a hydroxyl group at the C ¹¹ position of a steroid molecule is of great industrial importance, since it not only provides a change in the biological activity of the original molecule in the desired direction, but also creates the possibility for further modification of the molecule, for example, the introduction of a halogen atom at C ⁹ , in order to create pharmacologically more active derivatives. The implementation of 11 (α / β) -hydroxylation of steroids by microorganisms, especially progesterone, is economically important for the production of corticosteroids, since one of the main problems in their synthesis is the introduction of oxygen function in position 11, which is difficult to carry out by chemical methods. So, for example, the chemical synthesis of cortisone from bile acids includes ~ 30 stages and the total yield is several tenths of a percent [L. Fizer, M. Fizer. Steroids. Per. from English under the editorship of Doctor of Chemistry N.N.Suvorova and D.Sc. I.V. Trading. M .: Mir, 1964, p. 673]. On the contrary, the synthesis of cortisone acetate from 11α-hydroxyprogesterone consists of 10 stages and the total yield reaches 14.6% [SU 106380, 1956].

It is known that 11-hydroxylation of steroids in one stage with a high degree of selectivity is possible only biotechnologically using the enzyme system of microorganisms, mainly mycelial fungi, one of which is the genus Aspergillus. However, the species specificity of transformation of a number of pregnan steroids is inherent in this genus. Thus, the transformation of progesterone in a number of species (for example, A. terreus [K. Yildirim, A. Uzuner, and EY Gulcuoglu. Biotransformation of some steroids by Aspergillus terreus MRC 200365. Collect. Czech. Chem. Commun. 2010, Vol. 75 ( 6), 665-673], A. tamarii [K. Yildirim, A. Uzuner and EY Gulcuoglu. Baeyer-Villiger oxidation of some steroids by Aspergillus tamarii MRC 72400. Collect. Czech. Chem. Commun. 2011, Vol. 76 ( 6), 743-754], A. versicolor [HY Yang, HL Su, G. Du, GJ Shen, JX Sun, and HY Chen. Progesterone side-chain cleavage by Aspergillus versicolor. Advanced Materials Research. Advances in Chemical Engineering III 2013. Vols. 781-784, 1164-1167; Abed Nosrat M. Side chain degradation of progesterone by Aspergillus versicolor 79. Pakistan Journal of Biochemistry, 1972, Vol. 5 (1), 5-7], A. flavus [ ME Mostafa, and AA Zohri. Progesterone side-chain degradation by some species of Aspergillus flavus group. Folia Microbiol (Praha) . 2000; Vol. 45 (3), 243-7]) can proceed not in the direction of hydroxylation, but in the direction of elimination of the side pregnane chain with the formation of androstane compounds. Other strains of the genus Aspergillus are capable of hydroxylating progesterone by introducing a hydroxyl group at various positions of the molecule, including the 11α position. In this case, the direction of hydroxylation can significantly depend not only on the species affiliation of the strain, but also on the conditions of the transformation.

The biotransformation of progesterone with the formation of 11α-hydroxyprogesterone is best studied using Aspergillus ochraceus fungi, the selectivity of 11α-hydroxylation in which can reach 90% or more [T.K. Dutta, and T.V. Samanta. Bioconversion of progesterone by the activated immobilized conidia of Aspergillus ochraceus TS. Curr. Microbiol. 1999; Vol. 39 (6), 309-312; P. Somal, and C. L. Chopra. Microbial conversion of steroids. III: 11a-hydroxylation by fungal mycelium. Applied Microbiology and Biotechnology, 1985, Vol. 21 (5), 267-269]. Strains of A. ochraceus are used as industrial crops.

Aspergillus nidulans mycelium fungus is one of the most famous eukaryotic genetic systems and is widely used as a model system for deciphering cell cycle biology, pathogenicity, drug resistance, human diseases, primary and secondary metabolism of other microorganisms. Despite this, A. nidulans fungal strains as steroid-transforming microorganisms have been little studied. However, there are reports that this species has 11α-monooxygenase activity and is capable of transforming progesterone to form a predominantly 11α-hydroxy derivative. So, MJ Henry and HD Sisler [MJ Henry, and HD Sisler. Effects of sterol biosynthesis-inhibiting (SBI) fungicides on cytochrome P-450 oxygenations in fungi. Pesticide Biochemistry and Physiology, 1984, Vol. 22 (3), 262-275], studying the effect of fungicides that inhibit ergosterol biosynthesis in fungi on the cytochore-P450-dependent hydroxylation of progesterone, reported that A. nidulans (Eidam) Winter fungus (strain 003) hydroxylates progesterone at an initial concentration of 100 μg / ml to form a mixture containing 11α-hydroxyprogesterone (50%), 6β-hydroxyprogesterone (5%), 6β, 11α-dihydroxyprogesterone (14%) and unconverted progesterone (31%). In this case, lysis activity (side chain cleavage at the C ¹⁷ -C ²⁰ bond) was not observed in this strain.

It is also known that the culture of A. nidulans fungus (from the collection of the Culture Center of the Laboratory of Microbiological Chemistry, National Research Center, Cairo, local habitat), possessing steroid-11α-hydroxylating activity, can transform progesterone not only in 11α-hydroxyprogesterone, but also in 21-hydroxyprogesterone (aka 11-deoxycorticosterone) [A.N. El-Refai, and K.M. Ghanem. Some physiological relations of progesterone conversion by Aspergillus nidulans. Egyptian Journal of Microbiology, 1987, Vol. 22 (2), 327-338; A.H. El-Refai, and K.M. Ghanem. Microbial response to steroids. Egyptian Journal of Microbiology, 1989, Vol. 23 (1), 1-11]. The authors introduced progesterone into the growth medium in a solution of 96% ethanol with a final solvent concentration of 1%. Transformation of progesterone was carried out with a load of 1 g / l at a temperature of 30 ± 2 ° С on a shaker (200 rpm, amplitude 7 cm) for 48 hours. In this case, the formation of a dihydroxylated derivative - 6β, 11α-dihydroxyprogesterone was observed. It was concluded that at pH close to neutral, 6β-hydroxylation is a secondary process and 6β, 11α-dihydroxyprogesterone is formed from the initially formed 11α-hydroxyprogesterone [A.N. El-Refai, and K.M. Ghanem. Some physiological relations of progesterone conversion by Aspergillus nidulans. Egyptian Journal of Microbiology, 1987, Vol. 22 (2), 327-338].

, M. Tadra, and J.

. Microbial transformations of steroids XXIV. Separation of androstane 17-hydroxy-epimers. Folia Microbiol. 1964, Vol. 9(6), 380-382]. При этом отмечалось, что ацетилирование происходит только с 17β-гидроксипроизводным, тогда как соответствующий 17α-эпимер остается нетронутым. В этих условиях не происходит ацетилирования молекулы стероида с гидроксигруппой в положениях 11α, 11β, 20β и 21. Однако информация о способности мицелиальных грибов, к которым относится род Aspergillus, ацетилировать стероидные спирты, в литературных источниках отсутствует.It is known that microorganisms are capable of acetylating steroidal secondary alcohols. Thus, the ability of some species of yeast and yeast-like organisms (Saccharomyces fragilis, S. lactis, Candida pseudotropicalis and Torulopsis sphaerica) to acetylate testosterone is known [A.

, M. Tadra, and J.

. Microbial transformations of steroids XXIV. Separation of androstane 17-hydroxy-epimers. Folia Microbiol. 1964, Vol. 9 (6), 380-382]. It was noted that acetylation occurs only with the 17β-hydroxy derivative, while the corresponding 17α-epimer remains intact. Under these conditions, there is no acetylation of a steroid molecule with a hydroxy group at positions 11α, 11β, 20β and 21. However, there is no information on the ability of mycelial fungi, which include the genus Aspergillus, to sterilize steroid alcohols.

It is known from the prior art that the acetylation of 11α-hydroxyprogesterone is carried out chemically using acetic anhydride as the acetylating agent. So, the well-known chemical method [D.H. Peterson, N.S. Murray, S.H. Eppstein, L.M. Reineke, A. Weintraub, P.D. Meister, and H.M. Leigh. Microbiological Transformations of Steroids. I. Introduction of Oxygen at Carbon-11 of Progesterone. J. Am. Chem. Soc., 1952, Vol. 74 (23), 5933-5936] receive 11α-acetoxyprogesterone, acetylating 11α-hydroxyprogesterone (20 mg) with acetic anhydride (0.6 ml) in pyridine medium (0.6 ml). The mixture was incubated for 16 hours at room temperature. At the end of the reaction, the reaction mass is diluted with 25 ml of water, kept for 1 h, cooled to start crystallization. The crystals are separated by filtration, washed with water and dried. 16.1 mg of 11α-acetoxyprogesterone are obtained with a melting point of 176-177 ° C.

Thus, from the prior art it follows that the formation of 11α-acetoxyprogesterone during the transformation of progesterone by cultures of micellial fungi in general, in particular, the genus Aspergillus, specifically Aspergillus nidulans, was not previously observed. The use of A. nidulans fungi with steroid-11α-hydroxylating activity and capable of converting progesterone to 11α-hydroxyprogesterone to produce 11α-acetoxyprogesterone was reported for the first time. In this case, the esterification of 11α-hydroxyprogesterone with A. nidulans fungus with the formation of 11α-acetoxyprogesterone is the second stage of the progesterone transformation process and can be carried out either with or without 11α-hydroxyprogesterone from the culture fluid formed in the first stage of biotransformation.

11α-Hydroxyprogesterone (11α-hydroxypregn-4-en-3,20-dione, CAS No. 80-75-1) is a physiologically active derivative of progesterone (pregn-4-en-3,20-dione, CAS No. 57-83- 0) - a native steroid hormone synthesized by the corpus luteum of the ovary, the cortex (cortex) of the adrenal gland, seminal vesicles and placenta. Progesterone-based drugs are used as medicines in medicine and veterinary medicine. 11α-Hydroxyprogesterone, like its 11β-epimer, has inhibitory activity on the enzyme 11β-hydroxysteroid-dehydrogenase, which oxidizes the 11-hydroxy group to the 11-keto group, thus playing a regulatory role in maintaining electrolyte balance [G.W. Souness, S.A. Latif, J.L. Laurenzo, D.J. Morris 11 alpha- and 11 beta-hydroxyprogesterone, potent inhibitors of 11 beta-hydroxysteroid dehydrogenase (isoforms 1 and 2), confer marked mineralocorticoid activity on corticosterone in the ADX rat. Endocrinology. 1995, Vol. 136 (4), 1809-1812].

11α-Acetoxyprogesterone (11α-hydroxyprogesterone acetate, CAS No. 2268-98-6) has physiological activity, is an active pharmaceutical ingredient and refers to agents that reduce the risk of premature birth [http://www.imcopharma.cz/eng/api/11 -alfa-gidroksiprogesteron-acetat]. 11α-Acetoxyprogesterone also serves as a starting substrate in the synthesis of other steroid compounds.

11α-Acetoxyprogesterone and 11α-hydroxyprogesterone, being progesterone derivatives with progestational activity, can be used in combination with natural or synthetic estrogens as an active ingredient both in oral contraceptive preparations and in drugs for hormone replacement therapy of women in the postmenopausal period.

In addition, 11α-hydroxyprogesterone and its acetate can be used not only as progestogen agents, as described above, but also as antiandrogen agents to reduce sebaceous secretions in patients suffering from seborrhea, allopecia, and other skin diseases associated with hyperandrogenization [RU 2432952, 2011]. Also known are cosmetic compositions for hair and scalp care containing 11α-hydroxyprogesterone or 11α-acetoxyprogesterone [DE 2757024, 1979]. At the same time, it was noted that these compounds, when applied topically, have no hormonal side effects.

11α-hydroxyprogesterone and 11α-acetoxyprogesterone can be used as starting materials in the synthesis of other drugs for medical use. For example, 11α-hydroxyprogesterone can be used in the synthesis of 11-ketoprogesterone (ketogestin, CAS No. 516-15-4), which is then converted to cortisone or 11β-hydroxyprogesterone, a precursor of native hydrocortisone, by methods known in the art. The chemical method of transition from 11α-hydroxyprogesterone to 11-ketoprogesterone with a yield of more than 90% and further to 11β-hydroxyprogesterone is described in the patent [US 2015376225, 2015, examples 1 and 7, respectively]. In addition, 11α-hydroxyprogesterone can be used as a starting compound in the synthesis of promising analogues of the neurosteroid anesthetic alfaxolone [P.Y. Savechenkov, D.C. Chiara, R. Desai, A.T. Stern, X. Zhou, A.M. Ziemba, A.L. Szabo, Y. Zhang, J.B. Cohen, S.A. Forman, K.W. Miller, K.S. Bruzik. Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands. European Journal of Medicinal Chemistry. 2017. Vol. 136, P. 334-347].

Disclosure of invention

The technical problem solved by the present invention is the expansion of the range of microorganisms capable of carrying out 11α-hydroxylation of progesterone, namely, the creation of a new recombinant strain of ascomycete A. nidulans containing a new autonomously replicable plasmid capable of 11α-hydroxylation of progesterone, as well as expanding the assortment acetylation methods of the secondary hydroxyl group at the C ¹¹ atom of the progesterone molecule, due to the presence of the ability of the strain of the present invention to acetylate a secondary hydroxyl group at the C ¹¹ atom of the progesterone molecule, as an alternative to the chemical method of acetylation.

The problem was solved by using the A. nidulans strain 031 / pDHG25-SgrDI (pyrG ^- ) obtained by transforming the autonomously replicating plasmid pDHG25-SgrDI with the ampicillin resistance gene AP ^r , argB gene and the unique SgrDI restriction site into A. nidulans 0 strain31 ^- ; pyrG ^- ) - auxotroph of the wild-type strain A. nidulans VKPM F-1069 was not previously used for biotransformation of steroid compounds, including progesterone. Strain A. nidulans 031 (argB ^- ; pyrG ^- ) (synonym FP-308.1; AN031; CBS 129193) - auxotroph strain Aspergillus nidulans VKPM F-1069 (synonym FGSC A4; ATCC 3863, 12996, 26451; CBS 112.46; NRRL 194) carrying mutations argB2 (requiring arginine) and pyrG89 (requiring uridine and uracil).

Strain A. nidulans 031 / pDHG25-SgrDI (pyrG ^- ) is a new recombinant strain obtained for the first time, its steroid-11α-hydroxylating activity was detected for the first time. This strain was first used for biotransformation of progesterone. The ability of this strain to convert progesterone to 11α-hydroxyprogesterone and 11α-acetoxyprogesterone has been established.

To obtain A. nidulans strain 031 / pDHG25-SgrDI (pyrG ^- ), a new plasmid pDHG25-SgrDI was created. Plasmid pDHG25-SgrDI was constructed based on the known autonomously replicated plasmid pDHG25 for the organism Aspergillus nidulans [D. Gems, IL Johnstone, AJ Clutterbuck. An autonomously replicating plasmid transforms Aspergillus nidulans at high frequency. Gene, 1991, Vol. 98, 61-67] by introducing into it a unique SgrDI site using known molecular genetic methods [J. Sambrook, T. Maniatis, and EF Fritsch. Molecular cloning a laboratory manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; 1989].

To insert a unique SgrDI site, the known autonomous plasmid pDHG25 was restricted to the BamHI site using the BamHI restriction enzyme. Then, the ends of the fragment were dephosphorylated using alkaline phosphatase. The oligonucleotide linker Bam_SgrDI (SEQ ID NO: 1) was phosphorylated with T4 polynucleotide kinase and a dephosphorylated fragment with a linker was ligated using T4 DNA ligase.

The resulting plasmid pDHG25-SgrDI contains: replicon of plasmid pMB1; ampicillin resistance gene AP ^r , which provides replication in E. coli; argB gene A. nidulans; plot AMA1, providing autonomous replication in A. nidulans, which is a reverse repetition, which is the site of the centromere of chromosome A. nidulans. In FIG. 1 depicts a map of a ring autonomously replicated plasmid pDHG25-SgrDI.

The mutant strain A. nidulans 031 (argB ^- ; pyrG ^- ) was transformed with the obtained plasmid pDHG25-SgrDI carrying the argB gene, with selection on selective medium. Of the obtained transformants capable of growing on arginine-free medium, a strain designated A. nidulans 031 / pDHG25-SgrDI (pyrG ^- ) was selected for further work. [Aleksenko, AY, Makarova, NA, Nikolaev, IV, and Clutterbuck, AJ, Integrative and replicative transformation of Penicillium canescens with a heterologous nitrate-reductase gene, Curr. Genet., 1995, vol. 28, pp. 474-478].

The technical result is the creation of a new recombinant strain of mycelial fungus and its use for biocatalytic production of steroid alcohols and their esterification without the use of chemical methods. The preparation of 11α-acetoxyprogesterone by biotransformation of progesterone in one stage has advantages over the two-stage process known in the art, which includes the following steps: 1) microbiological 11α-hydroxylation of progesterone and 2) chemical acetylation of the secondary 11α-hydroxyl group. Biotechnological 11α-hydroxylation of progesterone and acetylation of the formed 11α-hydroxyprogesterone with or without isolation from the reaction mass is a cost-effective process, the advantages of which over the chemical method of acetylation are as follows:

- the process is carried out without the use of aggressive chemicals, such as acetic anhydride or acetic acid chloride, without the use of strong mineral acids as acetylation catalysts, such as 60% perchloric acid;

- biocatalytic esterification of 11α-hydroxyprogesterone is an alternative to the chemical method of acetylation, is an environmentally cleaner and safer method;

- biocatalytic acetylation proceeds regionally without the formation of a by-product of the Δ ⁴ -3-keto-enoacetyl group of the 11α-hydroxyprogesterone molecule - 3,11α-diacetoxypregn-3,5-diene-20-one, the content of which can be 20-30% using the chemical method of acetylation , which significantly complicates the purification of the target 11α-acetoxyprogesterone and leads to loss of its output.

To achieve the above technical result, it is proposed to use a new strain of Aspergillus nidulans according to the claimed invention as a biocatalyst for biotechnological processes of 11α-hydroxylation and 11α-acetoxylation of progesterone, as well as biocatalytic esterification of 11α-hydroxyprogesterone.

11α-Acetoxyprogesterone, if necessary, can be hydrolyzed in any convenient manner to form 11α-hydroxyprogesterone known in the art. For example, a chemical method is known for producing 11α-hydroxyprogesterone by solvolysis of 11α-acetoxyprogesterone by the action of (bistributyltin) oxide [MG Perez, and MS Maier. Mild deprotection of steroid esters by bis (tributyltin) oxide. Tetrahedron Letters, 1995. Vol. 36 (19), 3311-3314] or by the traditional method of chemical solvolysis under basic catalysis: in methanol in the presence of KOH [T. Kubota, and F. Hayashi. Studies on A-norsteroids - VI. Directing effects of C ₁₁ substituents on the addition of osmium tetroxide to steroidal Δ ^1,4 -3-ketones. Tetrahedron, 1967. Vol. 23, 995-1006].

Cultural and morphological features of the claimed strain.

Strain A. nidulans 031 / pDHG25-SgrDI (pyrG ^- ) on an agarized medium forms round colonies with a diameter of 30-35 mm after 7 days of growth. The surface is flat, convex, fluffy. Medium Density Texture. The edge of the colonies is dense, uneven. The color of the colonies in the germination zone is greenish-white. The reverse side is fawn brown. Exudate is absent. Characterized by intense sporulation. The color of the spores is bright green.

The main property of the strain according to the claimed invention is the presence of steroid-11α-hydroxylating activity and the ability to convert progesterone to 11α-acetoxyprogesterone, as well as acetylating activity and the ability to esterify 11α-hydroxyprogesterone.

The scheme for creating a new A. nidulans strain 031 / pDHG25-SgrDI (pyrG ^- ) of the present invention, based on the known A. nidulans VKPM F-1069 strain, is shown in FIG. 2.

In FIG. 3 presents a diagram of the transformation of progesterone by the claimed strain of Aspergillus nidulans 031 / pDHG25-SgrDI (pyrG ^- ).

Transformation of progesterone proceeds with the primary formation of 11α-hydroxyprogesterone. The acetylation of the hydroxyl group of the formed 11α-hydroxyprogesterone and its 6β-hydroxylation to form 6β, 11α-dihydroxyprogesterone are secondary, competitive transformation processes, and the biocatalytic esterification process has advantages. This conclusion is confirmed experimentally. With a 14-hour transformation duration by A. nidulans strain 031 / pDHG25-SgrDI (pyrG ^- ) in the culture medium, only 11α-acetoxyprogesterone and the untransformed starting substrate were detected. In addition, using 11α-hydroxyprogesterone instead of progesterone as the starting substrate under similar transformation conditions with A. nidulans strain 031 / pDHG25-SgrDI (pyrG ^- ), it was noted that exclusively 11α-acetoxyprogesterone was formed within 23 hours.

Thus, the essence of the claimed invention is to create a new recombinant strain of mycelial fungus A. nidulans 031 / pDHG25-SgrDI (pyrG ^- ) by transforming a new autonomously replicated plasmid pDHG25-SgrDI into A. nidulans strain 031 (argB ^- ; pyrGrof ^- ) - wild-type Aspergillus nidulans VKPM F-1069 - and its use for 11α-hydroxylation of progesterone and acetylation of 11α-hydroxyprogesterone with or without isolation of the latter from the culture fluid.

Advantages of using the A. nidulans strain 03l / pDHG25-SgrDI (pyrG ^- ) of the present invention are the ability to convert progesterone to 11α-hydroxyprogesterone and acetylate the hydroxyl group in the 11α-hydroxyprogesterone molecule to form 11α-acetoxyprogesterone as an 11α-hydroxyl culture medium is isolated without isolation, in situ.

Brief Description of the Drawings

In FIG. 1 depicts a map of a ring autonomously replicated plasmid pDHG25-SgrDI.

In FIG. Figure 2 shows the design of the strain Aspergillus nidulans 031 / pDHG25-SgrDI (pyrG) of the present invention, based on the known A. nidulans strain VKPM F-1069.

In FIG. Figure 3 shows the transformation of progesterone by A. nidulans strain 031 / pDHG25-SgrDI (pyrG).

In FIG. Figures 4, 7, and 15 show chromatograms of chromatography-mass spectrometric analysis of 11α-hydroxyprogesterone, 11α-acetoxyprogesterone, and 6β, 11α-dihydroxyprogesterone, respectively.

In FIG. 5 and 6 show the ¹ H NMR spectra of 11α-hydroxyprogesterone.

In FIG. Figures 8-14 show ¹ H NMR, ¹³ C NMR, DEPT-NMR, HSQC-NMR, and NMBC-NMR spectra of 11α-acetoxyprogesterone.

In FIG. 16-23 show ¹ H-NMR, ¹³ C-NMR, DEPT-NMR, HSQC-NMR and NMVC-NMR spectra of 6β, 11α-dihydroxyprogesterone.

The implementation of the invention

, and A.P. MacCabe; Consecutive gene deletions in Aspergillus nidulans: application of the Cre/loxP system. Curr Genet. 2006. Vol. 50, 217-224]); депонирован в коллекции "The CBS-KNAW culture collection" (Нидерланды) под номером CBS 129193 [http://www.westerdijkinstitute.nl/Collections/Biolomics.aspx?Table=CBS%20strain%20database].Auxotroph strain A. nidulans VKPM F-1069 - A. nidulans 031 (argB ^- , pyrG ^- ) (synonym FP-308.1 [U. Christensen, BS Gruben, S. Madrid, H. Mulder, I. Nikolaev, and RP de Vries , Unique Regulatory Mechanism for d-Galactose Utilization in Aspergillus nidulans, Appl Environ Microbiol. 2011 Vol. 77 (19), 7084-7087]; AN031 [JV Forment, D.

, and AP MacCabe; Consecutive gene deletions in Aspergillus nidulans: application of the Cre / loxP system. Curr Genet. 2006. Vol. 50, 217-224]); deposited in the collection "The CBS-KNAW culture collection" (Netherlands) under the number CBS 129193 [http://www.westerdijkinstitute.nl/Collections/Biolomics.aspx?Table=CBS%20strain%20database].

Progesterone (I) (CAS No. 57-83-0, C ₂₁ H ₃₀ O ₂ ), 11α-hydroxyprogesterone (II) (CAS No. 80-75-1, C ₂₁ H ₃₀ O ₃ ) and 11α-acetoxyprogesterone (C ₂₃ H ₃₂ O ₄ , Mw 372.5 are commercially available and can be purchased, for example, from Steraloids Inc. (USA) or from other manufacturers.

Inorganic salts were purchased from Fluka (Germany). Yeast extract and agar - Difco Becton Dickinson and company (Sparks, USA).

Other reagents, solvents, and inert gases are commercially available, were purchased from Russian manufacturers.

To prepare media for cultivation and transformation, aqueous solutions of acids, salts and alkalis, distilled water was used. Drinking tap water was used to rinse the extracts in organic solvents, unless otherwise specified.

All procedures, unless otherwise specified, were carried out at room temperature or ambient temperature, that is, in the range from 20 to 25 ° C. For processes requiring lower temperatures than room temperature, cooling was provided with cold tap water (in the range of 10 to 20 ° C), or a mixture of crushed ice and cold water (in the range of 5 to 10 ° C), or a mixture of crushed ice and calcium chloride (at a temperature below 5 ° C).

Microorganisms were cultured on a New Brunswick ™ Innova ^® 44 / 44R rocking chair in a thermostatically controlled room at a temperature of 37 ° C (240-250 rpm, amplitude 5 cm).

Labortechnik AG), при остаточном давлении 0,35±0,05 кгс/см² (35±5 кПа) и температуре воды в бане в диапазоне от 35-50°С в зависимости от природы упариваемого растворителя.Evaporation of solvents in vacuo was carried out using a Rotavapor (

Labortechnik AG), with a residual pressure of 0.35 ± 0.05 kgf / cm ² (35 ± 5 kPa) and a water temperature in the bath in the range from 35-50 ° C, depending on the nature of the evaporated solvent.

Drying of the product crystals to constant weight was carried out at a temperature of 35-45 ° C at atmospheric pressure or using a vacuum oven at a residual pressure of 0.35 ± 0.05 kgf / cm ² (35 ± 5 kPa).

A universal indicator paper with a range of values from 0 to 12 (Lahema, Czech Republic) was used to determine the pH of the washings.

Column chromatography was performed on a column (16 × 650 mm) using silica gel of the brand Silica gel 60 (0.040-0.063 mm) (Merck, Germany).

The elution was monitored by thin layer chromatography (TLC) using Silica gel 60 F254 plates (Merck, Germany) and solvent chromatographic systems: dichloromethane-acetone 9: 1 (v / v) or dichloromethane-acetone 4: 1 (v / v ) The plates were viewed in UV light at a wavelength of 254 nm, then they were sprayed with a 1% solution of vanillin in a 10% aqueous solution of HClO4, and developed at a temperature of 100-120 ° C.

The structure and purity of all isolated compounds was confirmed by at least one of the following methods: TLC (plates for TLC Silica gel 60 F254 (Merck, Germany)), mass spectrometry, elemental analysis, nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC).

Labortechnik AG).The melting point of the isolated compounds was determined on a device to determine the melting point of M-565

Labortechnik AG).

¹ H- and ¹³ C-NMR spectra were determined on a Bruker Avance-400 spectrometer (Bruker BioSpin GmbH) with an operating frequency of 400 MHz and 100.6 MHz, respectively, using deuterated chloroform (99.8% D, Sigma-Aldrich), or deuterated dimethyl sulfoxide (99.9% D, Sigma-Aldrich) as a solvent relative to tetramethylsilane (TMS NMR grade ≥99.9%, Sigma-Aldrich) as an internal standard, in parts per million (ppm).

High resolution mass spectra (HRMS) were recorded on a Bruker Daltonics micrOTOF-Q II instrument using electrospray ionization (ESI). The measurements were obtained in the mode of positive ions with the following parameters: capillary boundary voltage of 4500 V; mass range from m / z 50 to 3000; external calibration (Electricpray Calibrant Solution, Fluka); atomizer pressure 0.4 bar; flow rate 3 μl / min; nitrogen was used as dry gas (6 l / min); The interface temperature was set at 180 ° C. The sample was introduced into the chamber of a mass spectrometer from an Agilent 1260 HPLC system equipped with an Agilent Poroshell 120 EC-C18 column (3.0 × 50 mm; 2.7 μm) and protection using an autosampler. A sample was introduced in a solution of 50% acetonitrile (LC-MS qualification) in water (MilliQ ultrapure water, Merck Millipore KGaA, Germany), and the column was eluted with a mixture of acetonitrile (A) and water (B) in a concentration gradient with a flow rate of 400 μl / min in the following gradient parameters: 0-15% A for 6 min, 15% - 85% A for 1.5 min, 85% - 0% A for 0.1 min, 0% A for 2.4 min . The retention time was as follows: 11α-hydroxyprogesterone - 5.2 min; progesterone - 6.7 minutes; 11α-acetoxyprogesterone - 6.0 min; 6β, 11α dihydroxyprogesterone - 4.2 min.

The formation of 11α-acetoxyprogesterone as a result of the transformation of progesterone or 11α-hydroxyprogesterone by A. nidulans 031 / pDHG25-SgrDI (pyrG ^- ) fungus culture was confirmed by ¹ H NMR, ¹³ C NMR, 2D NMR spectra, and high resolution chromatography-mass spectrometry. In FIG. Nos. 5, 6, 7-14 and 16-23 show the spectra and in FIG. Nos. 4, 7 and 15 — chromatograms of the obtained compounds.

To confirm the formation of 11α-acetoxyprogesterone during the transformation of progesterone or 11α-hydroxyprogesterone by A. nidulans 031 / pDHG25-SgrDI (pyrG ^- ) culture, 11α-acetoxyprogesterone was obtained by chemical synthesis from 11α-hydroxyprogesterone using the acetylation method.

When carrying out the invention, in addition to the methods described in detail in the following examples, the methods well-known to those skilled in the art described in the manuals of molecular biology and genetic engineering were used [J. Sambrook, T. Maniatis, and E.F. Fritsch. Molecular cloning a laboratory manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; 1989; FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, and K. Struhl. Current Protocols in Molecular Biology, John Wiley and Sons, NY, 1997].

The yields of 11α-hydroxyprogesterone and 11α-acetoxyprogesterone obtained by biotransformation of progesterone and 11α-hydroxyprogesterone with the strain of the present invention and shown in the examples of the claimed invention are, of course, lower than those necessary for an efficient industrial process. However, examples are given only to illustrate the biocatalytic ability of the claimed strains to convert progesterone not only to 11α-hydroxyprogesterone, but also to 11α-hydroxyprogesterone acetate. It should be noted that the indicated acetylating ability was revealed by us for the first time. The yields can be significantly improved by optimizing the conditions for biotransformation in order to minimize the formation of 6β, 11α-dihydroxyprogesterone and shift the direction of secondary modification of 11α-hydroxyprogesterone towards acetylation. It is known that the composition of the products depends on the composition of the medium, the pH of the medium, the age of the culture, on the induction of the original substrate or product [A.N. El-Refai, and K.M. Ghanem Some physiological relations of progesterone conversion by Aspergillus nidulans. Egyptian Journal of Microbiology, 1987. Vol. 22 (2), 327-338]. Therefore, to reduce the activity of the secondary enzyme 6β-hydroxylase and to reduce the amount formed of 6β, 11α-dihydroxyprogesterone, it is necessary to optimize the parameters of the transformation process, for example, the pH of the medium, the age of the culture, the method of introducing the substrate, the composition of the medium for transformation, temperature conditions and other from the prior art. For example, Table 1 shows the effect of the composition of the medium on the conversion of progesterone by the claimed strain, on its 11α-monooxygenase activity (total) and on the relative selectivity of 11α-hydroxyprogesterone and 11α-acetoxyprogesterone, under other identical conditions.

For transformation, we used 2 media options of the following composition:

et al. [А.

, М. Tadra, J.

. Microbial transformations of steroids XXIV. Separation of androstane 17-hydroxy-epimers. Folia Microbiologica. 1964. Vol. 9(6), 380-382] для ацетилирования тестостерона дрожжами и описанная в статье А.Н. El-Refai и K.M. Ghanem [А.Н. El-Refai, and K.M. Ghanem. Some physiological relations of progesterone conversion by Aspergillus nidulans. Egyptian Journal of Microbiology, 1987. Vol. 22(2), 327-338] (среда II, рН 6,5), как наиболее благоприятная для конверсии прогестерона в 11α-гидрокси-производное культурой A. nidulans. Состав среды, г/л: глюкоза - 40; MgSO₄×7H₂O - 1; KH₂PO₄ - 0,74; пептон - 1; дрожжевой экстракт - 1; L-аспарагин - 0,7; уридин - 1,1; урацил - 1,2.1 - CM medium used by A.

et al. [BUT.

, M. Tadra, J.

. Microbial transformations of steroids XXIV. Separation of androstane 17-hydroxy-epimers. Folia Microbiologica. 1964. Vol. 9 (6), 380-382] for testosterone acetylation by yeast and described in the article by A.N. El-Refai and KM Ghanem [A.N. El-Refai, and KM Ghanem. Some physiological relations of progesterone conversion by Aspergillus nidulans. Egyptian Journal of Microbiology, 1987. Vol. 22 (2), 327-338] (medium II, pH 6.5), as the most favorable for the conversion of progesterone to the 11α-hydroxy derivative by A. nidulans culture. The composition of the medium, g / l: glucose - 40; MgSO ₄ × 7H ₂ O - 1; KH ₂ PO ₄ 0.74; peptone - 1; yeast extract - 1; L-asparagine - 0.7; uridine - 1.1; uracil - 1.2.

2 - minimal synthetic medium MM. The composition of the medium, g / l: glucose - 20; MgSO ₄ × 7H ₂ O - 0.52; KH ₂ PO ₄ - 1.52; NaNO ₃ - 0.85; CuSO ₄ × 5H ₂ O - 0.13; CaCl ₂ - 0.11; KCl - 0.52, H ₃ BO ₃ - 5 × 10 ^-6 ; MnSO ₄ × 5H ₂ O - 1 × 10 ^-3 ; ZnSO ₄ × 7H ₂ O - 0.8 × 10 ^-3 ; Na ₂ MoO ₄ × 4H ₂ O - 0.8 × 10 ^-3 ; FeSO ₄ × 5H ₂ O - 0.8 × 10 ^-3 ; uridine - 1.1; uracil - 1.2; 0.5 M phosphate-citrate buffer (pH 6.6) - 200 ml / l.

It is known that mineral salts added to the culture medium, in particular CuSO ₄ , can be steroid-C21 monooxygenase inhibitors (EC No. 1.14.99.10 [Springer Handbook of Enzymes, Vol. 27, Class 1. Oxidoreductases XII. EC 1.14 .15-1.97. Second edition. Springer. 2006, p. 302]). However, the experimental results showed that on the medium SM the formation of 21-hydroxyprogesterone as a transformation product is not observed. It should be noted that 21-hydroxyprogesterone along with 11α-hydroxyprogesterone is one of the progesterone transformation products in this medium by A. nidulans strain in a known manner [A.N. El-Refai, and KM Ghanem. Some physiological relations of progesterone conversion by Aspergillus nidulans. Egyptian Journal of Microbiology, 1987, Vol. 22 (2), 327-338].

The possibility of implementing the claimed invention is shown, but not limited, in examples of specific performance.

Example 1. Construction of the plasmid pDHG25-SgrDI

To insert a unique SgrDI site, the known autonomous plasmid pDHG25 was restricted to the BamHI site using the BamHI restrictase (Thermo Fisher Scientific, according to the manufacturer's instructions [https://www.thermofisher.com/order/catalog/product/ER0051]). Then, the ends of the fragment were dephosphorylated using alkaline phosphatase (Shrimp Alkaline Phosphatase from Thermo Fisher Scientific, according to the manufacturer's instructions [https://www.thermofisher.com/order/catalog/product/783901000UN]). The oligonucleotide linker Bam_SgrDI (SEQ ID No. 1) was phosphorylated with T4 polynucleotide kinase (SibEnzyme, according to the manufacturer's instructions [http://russia.sibenzyme.com/service/protocols/protocol17]) and with T4 DNA ligase (SibEnzyme, [http: //russia.sibenzyme.com/service/protocols/protocol13]) ligated the dephosphorylated fragment with a linker.

The following ligation conditions were used: a mixture consisting of 5 μl dephosphorylated fragment (500 ng), 10 μl linker, 1 μl 10-fold BamHI buffer (SibEnzyme), 2 μl 50-fold PEG400, 1 μl 10 mM ATP, 2 μl T4 DNA ligases (1 unit / μl, Sibenzyme) were incubated for 15 h at 14 ° С, then heated for 10 min at 65 ° С, cooled in ice.

5 μl of the obtained ligase mixture was used to transform ultra-competent cells of E. coli strain XL10Gold (Stratagene). Ampicillin-resistant clones were selected on LB medium with ampicillin. Additional selection was performed using restriction analysis of plasmid DNA preparations. For this, restriction was performed at the PstI, BglII sites, and a plasmid construct was selected that forms, after restriction, fragments of sizes 5272, 3954, 1578 nucleotides. The selected plasmid constructs were sequenced using the PrArgB_seq oligonucleotide (SEQ ID NO: 2). The selected construct (SEQ ID No. 3) was designated as pDHG25-SgrDI and used in further work to obtain A. nidulans strain 031 / pDHG25-SgrDI (pyrG ^- ).

Example 2. Obtaining a recombinant strain of Aspergillus nidulans 031 / pDHG25-SgrDI (pyrG ^- )

To obtain the recombinant strain A. nidulans 031 / pDHG25-SgrDI (pyrG ^- ), the strain A. nidulans 031 (argB ^- ; pyrG ^- ) was transformed with the plasmid pDHG25-SgrDI (obtained according to example 1), carrying the argB gene, with selection by marker argB2. [Aleksenko, AY, Makarova, NA, Nikolaev, IV, and Clutterbuck, AJ, Integrative and replicative transformation of Penicillium canescens with a heterologous nitrate-reductase gene, Curr. Genet., 1995, vol. 28, pp. 474-478]. A series of transformants was obtained from which strains capable of growing on medium without arginine were selected. The total cellular DNA of the transformants was isolated by the standard method, the presence of plasmids in the grown strains was confirmed by sequencing using the PrArgB_seq primer (SEQ ID No. 2). A clone carrying the pDHG25-SgrDI plasmid was selected. The selected strain was named A nidulans 031 / pDHG25-SgrDI (pyrG ^- ).

Example 3. The general method of biotransformation of progesterone by strain Aspergillus nidulans 031 / pDHG25-SgrDI (pyrG ^- ) on CM and MM for 66 hours

To obtain a spore suspension, the strain was grown on MPA agar medium (malt extract 3 g / l, peptone 1 g / l, agar 20 g / l, uridine 1.1 g / l, uracil 1.2 g / l) at a temperature of 37 ° C for 7-10 days. The spore suspension was plated in 750 ml rocking flasks containing 100 ml of culture medium, grown on a rocking chair (240-250 rpm, amplitude 5 cm) at a temperature of 37 ° C for 4 days. Then, progesterone was added to the growth medium in the form of a solution in dimethyl sulfoxide to a final concentration of 1 g / l, while the concentration of the solvent did not exceed 6% (vol.). Transformation was performed for 66 hours under the same conditions, while culturing strains without progesterone as a control in parallel. Each experiment was performed in triplicate.

To recover the progesterone transformation products, at the end of the incubation, the mycelium was filtered off, washed with dichloromethane on a filter. The culture fluid was extracted three times with equal volumes of dichloromethane. Steroids adsorbed on the mycelium were recovered by re-maceration (three times) using methanol and keeping without stirring for 4 hours. The methanol was evaporated, the residue containing water was extracted with dichloromethane three times. The combined extracts of the culture fluid and mycelium were washed with water, dried with Na ₂ SO ₄ , clarified with activated charcoal and evaporated until the cutoff ceased. The residue was dissolved in 5 ml of a mixture of ethyl acetate and dichloromethane (1: 2) and chromatographed on a column using 30 times the amount of silica gel to the weight of the residue after evaporation. A mixture of dichloromethane and acetone (0 to 25% acetone) was used as an eluent; TLC was used to control the elution. The chromatographic purity of the compounds was confirmed by TLC and NMR spectroscopy.

The results of the biotransformation of progesterone by A. nidulans strain 031 / pDHG25-SgrDI (pyrG ^- ) of the present invention are shown in table 1.

Example 4. Transformation of progesterone with a strain of Aspergillus nidulans 031 / pDHG25-SgrDI (pyrG ^- ) for 14 hours

A spore suspension of A. nidulans strain 031 / pDHG25-SgrDI (pyrG ^- ) was sown in 4 rocking flasks with a capacity of 750 ml each containing 100 ml of MM culture medium, grown on a rocking chair (240-250 rpm, amplitude 5 cm) at a temperature 37 ° C for 4 days. Then, progesterone in a dimethyl sulfoxide solution was added to the growth medium to a final concentration of 1 g / L, while the concentration of the solvent was 4% (vol.). Transformation was carried out for 14 hours under the same conditions.

To recover the progesterone transformation products, at the end of the incubation, the mycelium was filtered off, washed with dichloromethane on a filter. The culture fluid was extracted three times with equal volumes of dichloromethane. Steroids adsorbed on the mycelium were recovered by re-maceration (three times) using methanol and keeping without stirring for 4 hours. The methanol was evaporated, the residue containing water was extracted with dichloromethane three times. The combined extracts of the culture fluid and mycelium were washed with water, dried with Na ₂ SO ₄ , clarified with activated charcoal and evaporated until the cutoff ceased. The residue was dissolved in 5 ml of a mixture of ethyl acetate and dichloromethane (1: 2) and chromatographed on a column using 30 times the amount of silica gel to the weight of the residue after evaporation. A mixture of dichloromethane and acetone (0 to 25% acetone) was used as an eluent; TLC was used to control the elution. The eluate fractions of the same steroid composition were combined and evaporated to dryness. The crystallizing residue was triturated with ether, the precipitate was filtered off, dried to constant weight in a vacuum oven. The chromatographic purity of the compounds was confirmed by TLC and NMR spectroscopy.

Of 400 mg of progesterone loaded for transformation, 392 mg of progesterone (98%) and 5.82 mg of 11α-acetoxyprogesterone were obtained with a yield of 1.23% per loaded substrate (61.4% per converted substrate). According to TLC analysis, there are no other transformation products.

Example 5. Biocatalytic acetylation of 11α-hydroxyprogesterone with Aspergillus nidulans strain 031 / pDHG25-SgrDI (pyrG ^- )

A spore suspension of A. nidulans strain 031 / pDHG25-SgrDI (pyrG ^- ) was sown in 4 rocking flasks with a capacity of 750 ml each containing 100 ml of MM culture medium, grown on a rocking chair (240-250 rpm, amplitude 5 cm) at a temperature 37 ° C for 4 days. Then, 11α-hydroxyprogesterone in a dimethyl sulfoxide solution was added to the growth medium to a final concentration of 0.5 g / l, while the solvent concentration was 2% (vol.). Transformation was carried out for 23 hours under the same conditions.

To recover the transformation products of 11α-hydroxyprogesterone at the end of the incubation, the mycelium was filtered off, washed with dichloromethane on a filter. The culture fluid was extracted three times with equal volumes of dichloromethane. Steroids adsorbed on the mycelium were recovered by re-maceration (three times) using methanol and keeping without stirring for 4 hours. The methanol was evaporated, the residue containing water was extracted with dichloromethane three times. The combined extracts of the culture fluid and mycelium were washed with water, dried with Na ₂ SO ₄ , clarified with activated charcoal and evaporated until the cutoff ceased. The residue was dissolved in 5 ml of a mixture of ethyl acetate and dichloromethane (1: 2) and chromatographed on a column using 30 times the amount of silica gel to the weight of the residue after evaporation. A mixture of dichloromethane and acetone (from 0 to 20% acetone) was used as an eluent; TLC was used to control the elution. The eluate fractions of the same steroid composition were combined and evaporated to dryness. The crystallizing residue was triturated with ether, the precipitate was filtered off, dried to constant weight in a vacuum oven. The chromatographic purity of the compounds was confirmed by TLC and NMR spectroscopy.

From 200 mg of 11α-hydroxyprogesterone loaded for transformation, 168 mg of 11α-hydroxyprogesterone (84%) and 29.48 mg of 11α-acetoxyprogesterone were obtained with a yield of 13.08% per loaded substrate (81.73% per converted substrate). According to TLC analysis, there are no other transformation products.

The NMR spectra of the obtained 11α-acetoxyprogesterone are identical to the spectra of the standard sample and the spectra of the sample obtained by the chemical method of acetylation of 11α-hydroxyprogesterone.

Example 6. Obtaining 11α-acetoxyprogesterone by chemical method

To a solution of 34 mg of 11α-hydroxyprogesterone in 0.2 ml of acetic anhydride, 1 drop of 60% perchloric acid was added. The reaction mass was kept at room temperature for 1 h. At the end of the reaction, the reaction mass was added dropwise in 5 ml of water containing 1 ml of a 25% ammonia solution (pH ~ 7.5). It was stirred for 30 minutes, then the reaction mass was extracted with dichloromethane three times, the extract was washed with water until neutral, and evaporated to dryness. 39 mg of the residue was obtained, from which 27 mg of 11α-acetoxyprogesterone was recovered by preparative chromatography in 70.45% yield. Mp 173-174 ° C (lit. mp. 176-177 ° C [DH Peterson, N. S. Murray, SH Eppstein, LM Reineke, A. Weintraub, PD Meister, and HM Leigh. Microbiological Transformations of Steroids. I . Introduction of Oxygen at Carbon-11 of Progesterone. J. Am. Chem. Soc., 1952, Vol. 74 (23), 5933-5936]

Characterization of biotransformation products

11α-hydroxyprogesterone obtained by biotransformation of progesterone with Aspergillus nidulans strain 031 / pDHG25-SgrDI (pyrG ^- ).

Mp 164-166 ° C (lit. mp 164-165 ° C [K. Yildirim, and A. Kuru. Biotransformation of some steroids by Aspergillus candidus. Journal of Chemical Research, 2015. Vol. 39 (9), 546 -549]). M.w. 330.46.

High-resolution mass spectrum C ₂₁ H ₃₀ O ₃ (m / z): calculated for [M + H] ⁺ 331.2268, found 331.2264; calculated for [M + Na] ⁺ 353.2087, found 353.2088.

¹ H-NMR (400 MHz, CDCl ₃ ): 5.73 (s, 1H CH-4), 4.04 (ddd (pseudo-dt), 1H, ³ J _{11, 9} = 10.3 Hz, ³ J _{11, 12α} 4.8 Hz, ³ J _{11, 12β} 4.6 Hz, СН-11), 2.66 (dt, 1H, ² J _{1β, 1α} 13.7 Hz, ³ J _{1β, 2} 4.4 Hz, СН-1β), 2.55 (dd (pseudo-t) , 1H, ³ J _{17, 16α} 8.7 Hz, ³ J _{17, 16β} 9.1 Hz, СН-17), 2.48-2.26 (m, 5Н, СН ₂ -2 & СН ₂ -6 & СН-12β), 2.22-2.10 (m, 1H, CH-16β), 2.13 (s, 3H, CH ₃ -21), 2.02 (td, 1H, ² J _{1a, 1b} 13.7 Hz, ³ J _{1α, 2} 4.5 Hz, CH-1α), 1.87 -1.81 (m, 1H, СН-7β), 1.78-1.64 (m, 3Н, СН-8 & СН-15α & СН-16α), 1.58-1.47 (2Н (t, 1Н, ³ J _{12α, 12β} 11.3 Hz , СН-12α), 1.31 (s, 3Н, СН ₃ -19), 1.29-1.20 (m, 2Н, СН-12α & СН-14), 1.14 (t, 1Н, ³ J _{9, 8} 10.3 Hz, СН -9), 1.14-1.04 (2 × ddd, 2H, ³ J _{7α, 7β} 13.1 Hz, ³ J _{7, 6} 13.0 Hz, ³ J _{7α, 8} 3.8 Hz, CH ₂ -7α), 0.69 (s, 3H, CH ₃ -18).

11α-Acetoxyprogesterone obtained by biotransformation of progesterone with Aspergillus nidulans strain 031 / pDHG25-SgrDI (pyrG ^- ).

Mp 172-174 ° C (lit. mp. 176-177 ° C [DH Peterson, N. S. Murray, SH Eppstein, LM Reineke, A. Weintraub, PD Meister, and HM Leigh. Microbiological Transformations of Steroids. I . Introduction of Oxygen at Carbon-11 of Progesterone. J. Am. Chem. Soc., 1952, Vol. 74 (23), 5933-5936]. Mw 372.5;

High resolution mass spectrum C ₂₃ H ₃₂ O ₄ (m / z): calculated for [M + H] ⁺ 373.2373, found 373.2361; calculated for [M + Na] ⁺ 395.2193, found 395.2183.

¹ H-NMR (300 MHz, DMSO-d ₆ ): 5.67 (s, 1H CH-4), 5.14 (dt, 1H, ³ J _{11, 9} = 10.6 Hz, ³ J _{11, 10} 5.0 Hz, CH-11 ), 2.62 (dd (pseudo-t), 1H, ³ J _{17, 16α} 8.9 Hz, ³ J _{17, 16β} 9.1 Hz, СН-17), 2.47-2.35 (m, 2Н, СН-2β & СН- 6β), 2.29-2.12 (m, 3H, CH-6α & CH-12β & CH-2α), 2.10-1.96 (m, 1H, CH-16β), 2.05 (s, 3H, CH ₃ -21), 2.01 (s, 3H, CH ₃ -23), 1.91-1.78 (m, 3H, CH ₂ -1 & CH-7α), 1.72-1.60 (m, 3H, CH-16α & CH-15α & CH-8), 1.50 (t, 1H, ³ J _{12α, 12β} 11.6 Hz, СН-12α), 1.44 (t, 1H, ³ J _{9, 8} 10.6 Hz, СН-9), 1.39-1.29 (ddd, 1H, ³ J _{14, 8} 10.7 Hz, ³ J _{14, 15 β} 6.4 Hz, ³ J _{14, 15 α} 5.6 Hz, CH-14) 1.29 (br.s, 4H, CH ₃ -19 & CH-15β), 1.17-1.00 (2 × ddd, 2H, ³ J _{7α, 7β} 12.6 Hz, ³ J _{7, 6α} ~ ³ J _{7, 6β} 12.2 Hz, ³ J _{7α, 8} 3.1 Hz, CH ₂ -7), 0.64 (s, 3H, CH ₃ -18).

¹³ C-NMR (300 MHz, DMSO-d ₆ ): 208.61 (s, СО-20), 199.40 (s, СО-3), 170.14 (s, С-5), 170.10 (s, СО-22) 124.48 (s, СН-4), 70.58 (s, СНОН-11), 62.30 (s, СН-17), 55.34 (s, СН-9), 54.39 (s, СН-14), 45.02 (s, СН ₂ -12), 43.54 (s, С-13), 39.75 (s, С-10), 36.79 (s, СН ₂ -1), 34.72 (s, СН-8) 34.19 (s, СН ₂ -2), 33.03 (s, CH ₂ -6), 31.67 (s, CH ₂ -7), 31.37 (s, CH ₃ -21) 30.92 (s, CH ₃ -21), 24.16 (s, CH ₂ -15), 22.96 (s, CH ₂ -16), 22.02 (s, CH ₃ -21), 18.20 (s, CH ₃ -19), 14.24 (s, CH ₃ -18).

The spectra are identical to the spectra of a standard sample, as well as the spectra of a sample obtained by biocatalytic acetylation by strain A. nidulans 031 / pDHG25-SgrDI (pyrG ^- ), and a sample obtained by the chemical method of acetylation of 11α-hydroxyprogesterone

6β, 11α-dihydroxyprogesterone obtained by biotransformation of progesterone with Aspergillus nidulans strain 031 / pDHG25-SgrDI (pyrG ^- ).

Mp 242-244 ° C (lit. mp 244-246 ° C [Z. Habibi, M. Yousefi, Sh. Ghanian, M. Mohammadi, S. Ghasemi. Biotransformation of progesterone by Absidia griseolla var. Igachii and Rhizomucor pusillus Steroids, 2012, Vol. 77, p. 1446-1449]. Mw 346.46;

High resolution mass spectrum C ₂₃ H ₃₂ O ₄ (m / z): calculated for [M + H] ⁺ 373.2373, found 373.2361; calculated for [M + Na] ⁺ 395.2193, found 395.2183.

¹ H-NMR (300 MHz, DMSO-d ₆ ): 5.67 (s, 1H CH-4), 5.14 (dt, 1H, ³ J _{11, 9} = 10.6 Hz, ³ J _{11, 10} 5.0 Hz, CH-11 ), 2.62 (dd (pseudo-t), 1H, ³ J _{17, 16α} 8.9 Hz, ³ J _{17, 16β} 9.1 Hz, СН-17), 2.47-2.35 (m, 2Н, СН-2β & СН- 6β), 2.29-2.12 (m, 3H, CH-6α & CH-12β & CH-2α), 2.10-1.96 (m, 1H, CH-16β), 2.05 (s, 3H, CH ₃ -21), 2.01 (s, 3H, CH ₃ -23), 1.91-1.78 (m, 3H, CH ₂ -1 & CH-7α), 1.72-1.60 (m, 3H, CH-16α & CH-15α & CH-8), 1.50 (t, 1H, ³ J _{12α, 12β} 11.6 Hz, CH-12α), 1.44 (t, 1H, ³ J _{9, 8} 10.6 Hz, CH-9), 1.39-1.29 (ddd, 1H, ³ J _{14, 8} 10.7 Hz, ³ J _{14, 15 β} 6.4 Hz, ³ J _{14, 15 α} 5.6 Hz, CH-14) 1.29 (br.s, 4H, CH ₃ -19 & CH-15β), 1.17-1.00 (2 × ddd, 2H, ³ J _{7α, 7β} 12.6 Hz, ³ J _{7, 6α} ~ ³ J _{7, 6β} 12.2 Hz, ³ J _{7α, 8} 3.1 Hz, CH ₂ -7), 0.64 (s, 3H, CH ₃ -18).

¹³ C-NMR (300 MHz, DMSO-d ₆ ): 208.61 (s, СО-20), 199.40 (s, СО-3), 170.14 (s, С-5), 170.10 (s, СО-22) 124.48 (s, СН-4), 70.58 (s, СНОН-11), 62.30 (s, СН-17), 55.34 (s, СН-9), 54.39 (s, СН-14), 45.02 (s, СН ₂ -12), 43.54 (s, С-13), 39.75 (s, С-10), 36.79 (s, СН ₂ -1), 34.72 (s, СН-8) 34.19 (s, СН ₂ -2), 33.03 (s, CH ₂ -6), 31.67 (s, CH ₂ -7), 31.37 (s, CH ₃ -21) 30.92 (s, CH ₃ -21), 24.16 (s, CH ₂ -15), 22.96 (s, CH ₂ -16), 22.02 (s, CH ₃ -21), 18.20 (s, CH ₃ -19), 14.24 (s, CH ₃ -18).

Thus, the total selectivity of 11α-hydroxylation reaches 96% or more, and the content of 11α-acetoxyprogesterone in the product mixture exceeds 31%. The indicated ability of the inventive recombinant strain is confirmed not only by physicochemical methods for analyzing the structure of organic compounds (high resolution mass spectrometry, ¹ H, ¹³ C NMR and 2D NMR spectroscopy), but also by counter chemical synthesis.

Claims (3)

1. Recombinant strain of mycelial fungus A. nidulans 031 / pDHG25-SgrDI (pyrG ^- ), having steroid-11α-hydroxylating activity, the ability to convert progesterone to 11α-acetoxyprogesterone, acetylating activity and the ability to esterify 11α-hydroxyformer progesterone 031 (argB ^- ; pyrG ^- ) (AN031; CBS 129193) carrying the mutations argB2 and pyrG89, an autonomously replicated plasmid pDHG25-SgrDI containing the ampicillin resistance gene AP ^r , the argB gene and the unique SgrDI restriction site. 2. The recombinant strain according to claim 1, characterized in that A. nidulans 031 (argB ^- ; pyrG ^- ) (AN031; CBS 129193) is an auxotroph of the strain Aspergillus nidulans VKPM F-1069. 3. The use of the strain according to claim 1 for 11α-hydroxylation of progesterone with the formation of 11α-hydroxyprogesterone and for acetylation of 11α-hydroxyprogesterone to obtain 11α-acetoxyprogesterone.

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