Abstract Background Pregnenolone and progesterone are the life-important steroid hormones regulating essential vital functions in mammals, and widely used in different fields of medicine. Microbiological production of these compounds from sterols is based on the use of recombinant strains expressing the enzyme system cholesterol hydroxylase/C20-C22 lyase (CH/L) of mammalian steroidogenesis. However, the efficiency of the known recombinant strains is still low. New recombinant strains and combination approaches are now needed to produce these steroid hormones. Results Based on Mycolicibacterium smegmatis, a recombinant strain was created that expresses the steroidogenesis system (CYP11A1, adrenodoxin reductase, adrenodoxin) of the bovine adrenal cortex. The recombinant strain transformed cholesterol and phytosterol to form progesterone among the metabolites. When 3-methoxymethyl ethers of sterols were applied as bioconversion substrates, the corresponding 3-ethers of pregnenolone and dehydroepiandrosterone (DHEA) were identified as major metabolites. Under optimized conditions, the recombinant strain produced 85.2 ± 4.7 mol % 3-methoxymethyl-pregnenolone within 48 h, while production of 3-substituted DHEA was not detected. After the 3-methoxymethyl function was deprotected by acid hydrolysis, crystalline pregnenolone was isolated in high purity (over 98%, w/w). The structures of steroids were confirmed using TLC, HPLC, MS and 1H- and 13C-NMR analyses. Conclusion The use of mycolicybacteria as a microbial platform for the expression of systems at the initial stage of mammalian steroidogenesis ensures the production of valuable steroid hormones—progesterone and pregnenolone from cholesterol. Selective production of pregnenolone from cholesterol is ensured by the use of 3-substituted cholesterol as a substrate and optimization of the conditions for its bioconversion. The results open the prospects for the generation of the new microbial biocatalysts capable of effectively producing value-added steroid hormones.

BACKGROUND: Downstream processing is an important part of industrial steroid biotechnology. The traditional recovery methods require significant amounts of the organic solvents which are associated with environmental risks. There is a need for environmentally friendly downstream processing which also may open the prospects for regeneration and repeated use of the costly materials. RESULTS: Microbial conversion of phytosterol to C17-ketosteroids in methylated cyclodextrin (mCD) solutions was studied as a model bioprocess. Quantitative aspects of competitive complexation of mCD and natural beta-CD (beta-CD) with steroids were studied under different temperature modes and CD concentrations. The method of steroid recovery based on precipitation of insoluble beta-CD complexes ensured the complete isolation of androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione, 9a-hydroxyandrost-4-ene-3,17-dione, 20-hydroxymethyl-pregna-1,4-dien-3-one and 3 beta-hydroxyandrost-5-en-17-one from aqueous media. Analysis of the H-1 nuclear magnetic resonance (NMR) spectra showed a constant molar ratio of beta-CD to steroid equal to 2 in the obtained steroid beta-CD complexes. The extraction of the steroid from the beta-CD complex by small volumes of organic solvent provided a quantitative yield. The method developed ensures regeneration and repeated use of the mCDs without decrease of the basic bioconversion outputs, as was demonstrated in a series of phytosterol to androsta-1,4-diene-3,17-dione bioconversions by Mycolicibacterium neoaurum VKM Ac-1816D. CONCLUSIONS: Downstream processing based on manipulation of steroid solubility in aqueous cyclodextrin solutions is a promising alternative to traditional steroid extraction with organic solvents. This environmentally friendly approach ensures re-use of materials without decrease in bioconversion outputs.(C) 2023 Society of Chemical Industry (SCI).

Engineered mutants of Mycolicibacterium spp. are known producers of valuable steroid synthons with C19 or C22 skeleton. Here we describe a method for site-directed mutagenesis of Mycolicibacterium neoaurum strains, bioconversion from phytosterol, and selective purification of C23 steroid 24-norchol-4-ene-3,22-dione (24-NCED) and C22 steroid 20-hydroxymethylpregn-4-ene-3-one (20-HMP). The yields of crystalline products with 95% purity by the method here described are 2.74 ± 0.085 g for 24-NCED and 1.42 ± 0.085 g for 20-HMP from 10 g/L phytosterol. 20-HMP is recognized as the key precursor in chemical syntheses of pharmaceutical corticosteroids and 24-NCED is a promising synthon for the synthesis of valuable steroids and own potent biological activity.

BACKGROUND Laccase mediator systems (LMS) possess great potential in organic synthesis for selective oxidation in mild conditions. However, their usage in steroid synthetic chemistry is limited by low aqueous solubility of the substrates. In this study, the effect of steroid complexation with methylated cyclodextrin (mCD) on LMS oxidation has been studied. RESULTS LMS consisted of Trametes versicolor laccase and 1-hydroxybenzotriazole or TEMPO as redox mediators were used. The only product, - 3 beta-hydroxyandrost-5-en-7,17-dione (7-keto-DHEA) was detected during LMS oxidation of 3 beta-hydroxyandrost-5-en-17-one (DHEA) at substrate concentrations within solubility. The formation of mCD complex with DHEA led to a decrease in the concentration of the reaction form of the substrate and inhibited bioconversion. When DHEA concentrations over its aqueous solubility was used, 3 beta,7 alpha- and 3 beta,7 beta-dihydroxyandrost-5-en-17-one (7 alpha- and 7 beta-OH-DHEA) along with 7-keto-DHEA were formed. Both isomeric 7 alpha- and 7 beta-OH-DHEA were efficiently converted to 7-keto-DHEA by LMS in the presence of mCD. LMS-catalyzed oxidation of 20-hydroxymethylpregn-4-en-3-one (HMP) in the presence of mCD resulted in a single product, pregn-4-en-3-one-20-carbaldehyde (20-POA) and the reaction rate increased under elevated mCD concentrations. A preparative biochemical synthesis of 20-POA was carried out, which has not been so far reported. CONCLUSIONS mCD affects LMS-catalyzed oxidation of steroid alcohols. The mechanism includes formation of steroid-mCD inclusion complexes that could inhibit, or facilitate oxidation depending on the structure of steroid-CD complex and accessibility of the hydroxyl functions to the LMS action. (c) 2021 Society of Chemical Industry (SCI).

In this study, we developed a method for the production of androsta-9(11)-diene-3,17-dione (Δ9(11)-AD), which is a combination of phytosterol side-chain microbial oxidation with simultaneous 9α‑hydroxylation and subsequent chemical regio-selective dehydration of 9α-hydroxy-3,17-diketo-intermediate without isolation and purification. Phytosterol was converted into 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) with the use of the wild-type Mycobacterium sp. VKM Ac-1817D strain. The product was extracted from the culture medium with the use of an organic solvent and dehydrated in the extract with mineral acid. The resulting Δ9(11)-AD was purified with the selective crystallization method. Minor products were isolated and identified. It has been shown that this strain is capable of sterol transformation with the formation of methyl ester of 9α-hydroxypregn-4-ene-3-one-20-carboxylic acid. Our approach makes it possible to simplify the flow chart for production of the target compound. It not only eliminates 9-OH-AD loss but also minimizes the amount of production waste.

Microbiological synthesis of 7α- and 7β-hydroxy derivatives of testololactone and testolactone was developed based on bioconversion of dehydroepiandrosterone (DHEA) by fungus of Isaria fumosorosea VKM F-881 with subsequent modification of the obtained stereoisomers by actinobacteria. The first stage included obtaining of the stereoisomers of 3β,7(α/β)-dihydroxy-17a-oxa-D-homo-androst-5-en-17-ones in the preparative amounts. Then the conversion of 7-hydroxylated D-lactones obtained by selected actinobacteria of Nocardioides simplex VKM Ac-2033D, Saccharopolyspora hirsuta VKM Ac-666, and Streptomyces parvulus MTOC Ac-21v was studied. Under the transformation of 3β,7α-dihydroxy-17a-oxa-D-homo-androst-5-en-17-one and its corresponding 7β-stereoisomer by N. simplex VKM Ac-2033D and S. hirsuta VKM Ac-666 the 7α- and 7β-hydroxy-17a-oxa-D-homo-androst-4-ene-3,17-dione (7α- and 7β-hydroxytestololactone), 7α- and 7β-hydroxy-17a-oxa-D-homo-androsta-1,4-diene-3,17-dione (7α- and 7β-hydroxytestolactone) were obtained with molar yields in a range of 60.3–90.9 mol%. The crystalline products of 7α-hydroxytestololactone, 7α-hydroxytestolactone, and their corresponding 7β-hydroxy stereoisomers were isolated, and their structures were confirmed by mass spectrometry and 1 H-NMR spectroscopy analyses. The strain of Str. parvulus MTOC Ac-21v transformed 3β,7(α/β)-dihydroxy-17a-oxa-D-homo-androst-5-en-17-ones into the corresponding 3-keto-4-ene analogs and did not show 3-ketosteroid 1(2)-dehydrogenase activity. The activity of actinobacteria towards steroid D-lactones was hitherto unreported. The results contribute to the knowledge of metabolic versatility of actinobacteria capable of transforming steroid substrates and may be applied in the synthesis of potential aromatase inhibitors.

Two-step one-pot microbial transformation enables obtaining of valuable steroids that are difficult to produce chemically. Here we describe a method for obtaining 11α-hydroxyandrost-4-ene-3,17-dione (11α-HAD) from cheap and available natural sterols (phytosterols or cholesterol).11α-HAD is a primary adrenal steroid in mammals and also a key precursor in the syntheses of halogenated corticoids. Conventional routes for its obtaining are based on chemical synthesis, or microbial hydroxylation of androst-4-ene-3,17-dione (AD). AD in turn is produced primarily with microbial biotransformation of natural sterols by some actinobacteria.Consequent bioconversions of sterols using two microbial strains in one bioreactor vessel without separation and purification of AD provides high yield of 11α-HAD. At the first fermentation step, phytosterol is converted to AD with Mycobacterium neoaurum NRRL 3805B, or relative strains, to yield about 70% (mol/mol). At the second step, AD is almost fully (98%) hydroxylated at the position 11α with Aspergillus ochraceus VKM F-830, or other suitable organisms, in the same bioreactor. At the average, 30% (w/w) of the high-purity crystalline 11α-HAD can be obtained.The method can be exploited for production of 11α-HAD for practical use.

Laccase mediator systems (LMS) were studied as catalysts for steroid oxidation. The fungal lac cases from Lentinus strigosus 1566 and Trametes versicolor were used in the work. Among five mediators screened, 1-hydroxybenzotriasol (HBT) excelled in activity. The LMS effectively catalyzed oxidation of 3 beta-hydroxy-Delta(5)-steroids like DHEA (3 beta-hydroxyandrost-5-en-17-one) and pregnenolone (3 beta-hydroxypregn-5-en-20-one), while no activity was observed towards 3-oxo-4-ene-steroids (androstenedione, 9 alpha-hydroxyandrostenedione, testosterone and 20-hydroxymethylpregn-4-en-3-one). The pathway of DHEA oxidation by LMS included the hydroxylation at positions 7 alpha and 7 beta followed by oxidation of the corresponding 7(alpha/beta)-alcohols to form 3 beta-hydroxyandrost-5-ene-7,17-dione. Regiospecific oxidation of allylic hydroxyl functions by LMS was confirmed using 3 beta,7 alpha and 3 beta,7 beta-dihydroxyandrost-5-en-17-ones as substrates. 3 beta-Hydroxypregn-5-ene-7,20-dione was produced with LMS as an only product from pregnenolone. The yield of crystalline product reached 58.3% yield with a purity of 96%.The results demonstrate that application of LMS may be a promising approach for steroid oxyfunctionalization. (C) 2015 Elsevier B.V. All rights reserved.

La luxation congénitale du genou (LCG) est une entité rare, dont la séméiologie clinique à la naissance n’est pas toujours bien analysée. Les classifications proposées ne permettent pas d’orienter les choix thérapeutiques. Le but de cette étude était de proposer une classification des LCG en période néonatale.Une classification fondée sur la sévérité des signes cliniques en période néonatale était aisément identifiable sur des critères simples.Cinquante et une LCG (40 patients) examinées en période néonatale ont été incluses. Les caractéristiques de la réduction et sa stabilité permettaient de distinguer 3 types. Le type I correspondait à des LCG réductibles sans difficulté avec un ressaut de réduction lors du passage des condyles fémoraux lors de la flexion et stables en flexion. Le type II incluait des luxations « récalcitrantes », c’est-à-dire réductibles avec un « piston » postéro-antérieur, mais instables avec luxation itérative dès que l’appui postéro-antérieur sur les condyles fémoraux était relâché. Le type III désignait des luxations irréductibles. Le nombre de sillons cutanés antérieurs, l’amplitude articulaire (AA) globale, le déficit de flexion et la stabilité de la réduction étaient notés pour chacun des types.L’âge moyen à la première consultation était 5,6 jours (0 à 30). Les LCG étaient de type I, II et III dans respectivement 28, 16 et 7 cas. Le nombre de sillons cutanés, la flexion et les AA initiales étaient plus élevés dans le type I que dans les types II et III.Cette classification clinique en période néonatale est originale, logique et simple. Elle peut revêtir un intérêt pronostique et orienter le traitement.Niveau IV. Série rétrospective monocentrique.

Селективная микробиологическая конверсия растительных стеринов позволяет получить ключевой интермедиат синтеза жизненно важных стероидных фармацевтических субстанций группы глюкокортикоидов – -гидроксиандрост-4-ен-3,17-дион (9-ОН-АД). В настоящей работе показано, что в качестве субстратов для микробиологической трансформации можно использовать обогащенные стеринами промышленные отходы переработки растительного сырья, а именно неомыляемый остаток таллового пека и его производные. Ряд простых и эффективных 1–2-стадийных методов фракционирования позволил получить производные, содержащие 51–88% (по массе) трансформируемых стеринов. Проведен сравнительный анализ динамики процессов биоконверсии полученных стеринобогащенных фракций таллового пека и коммерческого фитостерина. Показано, что биоконверсия ряда обогащенных стеринами образцов при скорости накопления продукта, в 2–2.5 раза меньшей, чем в контрольном образце (коммерческий фитостерин), приводит к достижению приемлемых значений мольного выхода 9-ОН-АД в интервале 53–57%. Полученные результаты позволяют сократить технологический путь от первичных продуктов переработки возобновляемого сырья (сопутствующие продукты переработки древесины) до целевых стероидных фармацевтических субстанций, получаемых из 9-ОН-АД.

The transformation of dehydroepiandrosterone by Spicaria fumoso-rosea VKM F-881 produced 7α- and 7β-hydroxy-dehydroepiandrosterone, 3β,7α-dihydroxy-17a-oxa-D-homo-androst-5-en-17-one, and 3β,7β-dihydroxy-17a-oxa-D-homo-androst-5-en-17-one. The yield of the main product—3β,7β-dihydroxy-17a-oxa-D-homo-androst-5-en-17-one—was 49.5–72 mol % at substrate loadings of 5–20 g/L. Lactone formation proceeded through 7α- and 7β-hydroxy derivatives of dehydroepiandrosterone. The structure of the products was determined by mass spectrometry, 1H-NMR spectroscopy, and 13C-NMR spectroscopy. The proposed microbiological method for producing steroid lactones opens prospects for the synthesis of novel steroid compounds.

Selective microbial conversion of plant sterols allows direct production of 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD), a key intermediate in the synthesis of important pharmaceutical substances from the steroid glucocorticoid group. As substrates for bioconversion, sterol-enriched wastes of local pulp-and-paper industry, particularly, unsaponifiable tall pitch residue and its derivatives can be used. A series of simple and effective one- or two-stage fractionation procedures yielded the derivatives containing 51–88 wt % of convertible sterols. The analysis of the bioconversion dynamics of the sterol-enriched samples demonstrated that while the 9-OH-AD accumulation rates were 2–2.5 times lower than with commercial phytosterol, acceptable molar yields of 53–57% of the product were obtained. The obtained results make it possible to optimize the technological chain starting from the primary products of processing of renewable raw materials (by-products of wood processing) and ending with the desired steroid pharmaceutical substances via 9-OH-AD.

Molecular-imprinting by cross-linking of ligands of β-cyclodextrin (CD) complex with steroids has been developed for the synthesis of tailor-made CD dimer. Steroids of androstane (9α-hydroxy-androst-4-en-3,17-dione, androst-4-en-3,17-dione, androsta-1,4-dien-3,17-dione (ADD)) and pregnane (hydrocortisone, 6-methyl-hydrocortisone, 20-hydroxymethylpregna-1,4-diene-3-one (HMPD)) series were used as template molecules. For imprinting procedure, crystalline β-CD complexes of exact stoichiometry (β-CD:steroid template = 2:1) were synthesized following by toluene 2,4-diisocyanate (TDI) cross-linking. The attempts to produce CD dimer for steroid without hydrophobic side chain failed, while tailor-made CD dimer has been obtained using HMPD as a template. The dimer was characterized by 1H NMR and mass-spectrometry. The complex stability constant (KS) towards HMPD template exceeded 107 M−1. The KS of CD dimer with ADD exceeded the corresponded value of TDI-modified CD monomer by more than an order of magnitude. The dimer was applied for quantitative extraction of ADD from aqueous solution using dialysis membranes impermeable for CD. The value of KS for ADD estimated from balanced concentrations of dialysis data corresponded to that calculated by nonlinear spectrometric method.

The synthesis of 3beta-hydroxy-androsta-5,7-dien-17-one from 3beta-hydroxy-androst-5-en-17-one (dehydroepiandrosterone, DHEA) via microbial 7alpha-hydroxylation has been accomplished. At the first stage, 3beta,7alpha-dihydroxy-androst-5-en-17-one was obtained in high yield (71.2%) using a strain of Gibberella zeae VKM F-2600, which was first applied for DHEA conversion. The further route included the substitution of 7alpha-hydroxyl group with chlorine followed by a dehydrochlorination stage, and required minimal purifications of the intermediate products. The steroids obtained at every step were characterized by TLC,1H NMR, MS, UV- and IR-spectrometry. The combination of microbial and chemical steps ensured 54.6% yield of the target 3beta-hydroxy-androsta-5,7-dien-17-one from DHEA and can be applied for obtaining novel vitamin D derivatives.

The inhibitory effect of methylated ss-cyclodextrin ( mCD) on steroid degradation was studied using the degradation of 9 alpha-hydroxyandrost4- ene-3,17-dione (9-OH-AD) by Mycobacterium sp. VKM Ac-1817D as a model process. The formation of the [9-OH-AD-mCD] complex was shown by 1 H NMR-spectroscopy. The biodegradation of 9-OH-AD by whole and disrupted cells was carried out at 30 degrees C in aqueous solutions with or without mCD. Enzyme kinetic parameters were calculated by non-linear regression of the Michaelis-Menten plot. The complexation of 9-OH-AD and mCD was evaluated via the stability constant for the [9-OH-AD-mCD] complex. The V-max and K-M values calculated for the free (noncomplex) steroid in mCD solutions corresponded to steroid degradation in the absence of mCD. The inclusion complex [9-OH-AD-mCD] was shown to be resistant to enzymatic degradation. The inference is made that the "guest-host'' molecular complexation with cyclodextrin can be used for the control of steroid bioconversions.