In this paper, we present an efficient procedure for the transformation of a soybean phytosterol mixture into pregna-1,4,16-triene-3,20-dione, which is a key intermediate for the synthesis of valuable corticoids. The possibility of using two alternative methods for 1(2)-dehydrogenation of pregna-4,16-diene-3,20-dione - chemical and microbiological - was shown. Microbiological 3-keto-4-ene steroid 1(2)-dehydrogenation was carried out using actinobacterial cells of Nocardioides simplex VKM Ac-2033D. The structures of the synthesized compounds were confirmed by the IR, MS, and 1H-NMR methods.

Molecular mechanisms of C19-steroid core degradation have been intensively studied mostly in mycolic acid rich actinobacteria, mainly in the representatives of Mycobacterium, Mycolicibacterium and Rhodococcus genera, whilst much less data evidencing functionality of the 9(10)-seco pathway in other actinobacteria was reported. In this study, degradation of androstenedione (AD), androstadienedione (ADD), testosterone (T), Δ1-dehydrotestosterone (DT) and 9α-hydroxyandrostenedione (9α-OH-AD) by the biotechnologically relevant actinobacterium of Nocardioides simplex VKM Ac-2033D was investigated. Key intermediates of 9α-OH-AD degradation were isolated and identified as 3-hydroxy-9,10-seco-androsta-1,3,5(10)-triene-9,17-dione (3-HSA) and 3,17β-dihydroxy-9,10-seco-androsta-1,3,5(10)-trien-9-one (3,17-DHSA). The structures of the compounds were confirmed by MS, 1H- and 13C-NMR. Differential gene expression on medium with glycerol and yeast extract with and without AD addition was estimated. The expression of two sets of the genes related to the 9(10)-seco pathway was increased in the presence of AD. One set comprised the genes from the KstR2-regulons in the clusters A and C, while another set included the genes without the binding sites for KstR/KstR2 (Cluster D). These genes putatively encoded 3-oxosteroid-Δ1-dehydrogenase and 3-oxosteroid 9α-hydroxylase, as well as a group of enzymes dealing with the ring B opening (HsaA3, HsaB3, HsaC3 and HsaD3). Process of degradation of exogenous C19-steroids in N. simplex proceeds via the 9(10)-seco pathway and can be controlled not only by KstR2 regulator, but also by other transcriptional factors. The results contribute to the knowledge on steroid core degradation in actinobacteria and are of significance at the development of methods for production of valuable indane compounds.

17β-Hydroxysteroid dehydrogenase (17β-HSD) is an enzyme used in biotechnology for producing testosterone from phytosterol. Heterologous 17β-HSD from the fungus Cochliobolus lunatus catalyzes NADPH-dependent reduction of the 17-oxo group of androstenedione/androstadienedione formed in mycolicibacterial cells as a result of the inherent polyenzymatic process of side chain oxidation of phytosterols, yielding testosterone/Δ1-dehydrotestosterone, respectively. The object of this study was heterologous 17β-HSD from the fungus C. lunatus (17β-HSDCl) with a 6×His tag (6×His-17β-HSDCl), synthesized in the cells of actinobacteria Mycolicibacterium neoaurum. Isolation and purification of the recombinant enzyme were performed using affinity chromatography. The 6×His-17β-HSDCl enzyme preparation exhibited the highest activity toward androstenedione. Activity of the 6×His-17β-HSDCl depended on NADPH and was observed in the pH range from 6.0 to 9.0 with an optimum at pH 7.0. Analysis of kinetic characteristics showed that the properties of the heterologous enzyme 6×His-17β-HSDCl synthesized in M. neoaurum cells are comparable with those reported for the 17β-HSD enzyme isolated from the fungus C. lunatus, as well as for the recombinant 17β-HSDCl enzymes synthesized in Escherichia coli and Mycolicibacterium smegmatis cells. The results expand our knowledge on microbial 17β-HSDs and suggest potential for the use of the recombinant M. neoaurum strains expressing a codon-optimized cDNA sequence encoding 17β-HSDCl from the fungus C. lunatus for producing testosterone from phytosterol.

A plasmid containing the genes of a fungal 17β-hydroxysteroid dehydrogenase, which catalyzes the reduction of the steroid core at the C17 position, and mycobacterial glucose-6-phosphate dehydrogenase, which promotes the recycling of the essential coenzyme NAD(P)H, was constructed. Its constitutive expression in well-studied Mycolicibacterium neoaurum strains made it possible to increase significantly the yield of C-17 hydroxysteroids. In particular, recombinant strains created on the basis of M. neoaurum VKM Ac-1815D and M. neoaurum NRRL B-3805 ΔkstD exhibited predominant accumulation of testosterone, while the strain based on M. neoaurum VKM Ac-1816D accumulated dehydrotestosterone and testosterone simultaneously.

Cholesterol determination by cholesterol oxidase reaction is a fast, convenient, and highly specific approach with widespread use in clinical diagnostics. Routinely, endpoint measurements with 4-aminophenazone or 4-aminoantipyrine as chromogens and sodium cholate, surfactants, or alcohols as solubilizing agents are used. Here we describe a novel kinetic method to determine cholesterol in 0.05-0.75 mM range in neutral or acidic buffers by use of recombinant cholesterol oxidase from Nocardioides simplex in a coupled reaction with horseradish peroxidase, ABTS as a chromogen, and methyl-β-cyclodextrin as a solubilizing agent.

Steroids are abundant molecules in nature, and various microorganisms evolved to utilize steroids. Thermophilic actinobacteria play an important role in such processes. However, very few thermophiles have so far been reported capable of degrading or modifying natural sterols. Recently, genes putatively involved in the sterol catabolic pathway have been revealed in the moderately thermophilic actinobacterium Saccharopolyspora hirsuta VKM Ac-666T, but peculiarities of strain activity toward sterols are still poorly understood. S. hirsuta catalyzed cholesterol bioconversion at a rate significantly inferior to that observed for mesophilic actinobacteria (mycobacteria and rhodococci). Several genes related to different stages of steroid catabolism increased their expression in response to cholesterol as was shown by transcriptomic studies and verified by RT–qPCR. Sequential activation of genes related to the initial step of cholesterol side chain oxidation (cyp125) and later steps of steroid core degradation (kstD3, kshA, ipdF, and fadE30) was demonstrated for the first time. The activation correlates with a low cholesterol conversion rate and intermediate accumulation by the strain. The transcriptomic analyses revealed that the genes involved in sterol catabolism are linked functionally, but not transcriptionally. The results contribute to the knowledge on steroid catabolism in thermophilic actinobacteria and could be used at the engineering of microbial catalysts.

Mycolicibacterium smegmatis mc2155 has been genetically modified to be used as a platform for the expression of foreign cytochrome P450 monooxygenases by introducing deletions in the kshB and kstD genes that encode key stages of the enzymatic destruction of the steroid nucleus. Three sets of genetic constructs have been created for heterologous expression of the genes of cytochromes P450 CYP106A1 from Bacillus megaterium DSM319 and CYP106A2 from Bacillus megaterium ATCC13368 in Mycolicibacterium smegmatis mc2155 (ΔkshBΔkstD) cells. The recombinant plasmids contained monocistronic expression cassettes of cytochrome genes (NS31 and pNS32), or tricistronic cassettes of cytochrome genes together with cDNA copies of adrenodoxin and andrenodoxin reductase genes of the bovine adrenal cortex (pNS33 and pNS34), or monocistronic gene cassettes of chimeric cytochromes fused with the DNA sequence encoding the CYP116B2 reductase domain from the soil bacterium Rhodococcus sp. NCIMB 9784 (pNS35 and pNS36). The recombinant strains of mycolicibacteria were shown to selectively monohydroxylate androstenedione (AD) under growth conditions. The product was identified as 15-hydroxyandrostenedione (15-OH-AD) by mass spectrometry and 1H and 13C NMR spectroscopy. The maximum level of 15-OH-AD production (17.3 ± 1.5 mg/L) was observed when using the recombinant M. smegmatis mc2155 (ΔkshBΔkstD) (pNS32) strain, which expresses a single cyp106A2 gene from B. megaterium ATCC13368. Host proteins of M. smegmatis mc2155 were shown to be capable of supplying electrons to heterologous cytochromes to support their hydroxylating activity. The results are of priority character, expand the understanding of the hydroxylation of steroid compounds by bacterial cytochromes CYP106A1/A2 and are important for the creation of microbial strains producing valuable hydroxysteroids. cyp106A1, cyp106A2, cytochrome P450, heterologous expression, Bacillus megaterium, Mycolicibacterium smegmatis, 15β-hydroxylation, bioconversion, steroids This work was supported by the Russian Science Foundation (project No. 21-64-00024).

Изучена эффективность метилглюкамина акридонацетата у детей дошкольного и школьного возраста, страдающих хронической Эпштейна — Барр вирусной инфекцией. Больных рандомизировали в группы стандартной терапии (n = 30) и стандартной терапии + метилглюкамина акридонацетат (n = 30) внутримышечно, 10 мг/кг по схеме: 1, 2, 4, 6, 8, 11, 14, 17, 20, 23 дни. В результате применения метилглюкамина акридонацетата отмечена более низкая частота эпизодов острых респираторных инфекций до 3,3 ± 1,2 раз в год, (p < 0,05), что приводило к уменьшению показателей инфекционного индекса до 1,2 ± 0,2 (p < 0,05) и индексу острой заболеваемости до 0,2 ± 0,2 (p < 0,05), по сравнению со стандартной терапией. Кроме того, в группе метилглюкамина акридонацетата зафиксировано снижение частоты интоксикационного на 29 % (2,7 ± 0,2 сут в сравнении с 3,8 ± 0,4 сут в группе 1, p < 0,05), лимфопролиферативного на 27,2 % (во группе 2 – 8,3 ± 0,64 сут в сравнении с 11,4 ± 0,47 сут в группе 1, p < 0,01), артралгического на 20,6 % (в группе 2 – 2,7 ± 0,34 сут против 3,4 ± 0,28 сут в группе 1, p < 0,05), кардиального на 26,4 % (в группе 2 – 3,9 ± 0,40 сут в сравнении с 5,3 ± 0,46 сут в группе 1, p < 0,05), вегетовисцерального — на 37,8 % (в группе 2 – 8,4 ± 0,40 сут в сравнении с 13,5 ± 0,35 сут в группе 1, p < 0,01) и гастроинтестинального синдромов — на 26,4 % (в группе 2 – 5,3 ± 0,52 сут в сравнении с 7,2 ± 0,82 сут в группе 1, p < 0,05), регистрировалось более быстрое исчезновение серологических маркеров репликации вируса Эпштейна – Барр (p < 0,05). Нежелательные явления при назначении метилглюкамина акридонацетата не отмечены. Таким образом, установлена высокая эффективность и безопасность применения метилглюкамина акридонацетата при активной форме хронической Эпштейна – Барр вирусной инфекции у детей.

Cholesterol oxidase is a highly demanded enzyme used in medicine, pharmacy, agriculture, chemistry, and biotechnology. It catalyzes oxidation of 3β-hydroxy-5-ene- to 3-keto-4-ene- steroids with the formation of hydrogen peroxide. Here, we expressed 6xHis-tagged mature form of the extracellular cholesterol oxidase (ChO) from the actinobacterium Nocardioides simplex VKM Ac-2033D (55.6 kDa) in Escherichia coli cells. The recombinant enzyme (ChONs) was purified using affinity chromatography. ChONs proved to be functional towards cholesterol, cholestanol, phytosterol, pregnenolone, and dehydroepiandrosterone. Its activity depended on the structure and length of the aliphatic side chain at C17 atom of the steroid nucleus and was lower with pregnenolone and dehydroepiandrosterone. The enzyme was active in a pH range of 5.25÷6.5 with the pH optimum at 6.0. Kinetic assays and storage stability tests demonstrated that the characteristics of ChONs were generally comparable with or superior to those of commercial ChO from Streptomyces hygroscopicus (ChOSh). The results contribute to the knowledge on microbial ChOs and evidence that ChO from N. simplex VKM Ac-2033D is a promising agent for further applications.

— New methods for testosterone production from phytosterol were developed based on its cascade two-stage transformation by actinobacteria Mycolicibacterium neoaurum VKM Ac-1815D and Nocardioides simplex VKM Ac-2033D. Efficient oxidation of the phytosterol side chain by M. neoaurum resulted in formation of the main and side products: androst-4-en-3,20-dione (AD) and androsta-1,4-dien-3,20-dione (ADD), respectively, which were subsequently converted to testosterone by N. simplex . The latter reaction was reversible and catalyzed by the membrane-associated 17β-hydroxysteroid dehydrogenase (17β-HSD) capable of both oxidation and reduction of androstendione at C17. Addition of glucose and limited aeration were found to be the key factors providing for a shift of the 17β-HSD activity towards reduction in whole N. simplex cells. Testosterone production from phytosterol was realized using two approaches: (i) based on M. neoaurum cells inactivation after phytosterol conversion and application of the resting N. simplex biomass for androstenedione reduction and (ii) based on sequential application of the two living cultures. Under optimized conditions, the total yield of testosterone from phytosterol (10 g/L) reached 53 mol %. The results exceeded those reported so far for cascade phytosterol bioconversion to testosterone and may be used as a basis for development of new biotechnologies for production of the valuable steroid compounds, intermediates in the synthesis of modern medical preparations .

The application of thermophilic microorganisms opens new prospects in steroid biotechnology, but little is known to date on steroid catabolism by thermophilic strains. The thermophilic strain Saccharopolyspora hirsuta VKM Ac-666T has been shown to convert various steroids and to fully degrade cholesterol. Cholest-4-en-3-one, cholesta-1,4-dien-3-one, 26-hydroxycholest-4-en-3-one, 3-oxo-cholest-4-en-26-oic acid, 3-oxo-cholesta-1,4-dien-26-oic acid, 26-hydroxycholesterol, 3β-hydroxy-cholest-5-en-26-oic acid were identified as intermediates in cholesterol oxidation. The structures were confirmed by 1H and 13C-NMR analyses. Aliphatic side chain hydroxylation at C26 and the A-ring modification at C3, which are putatively catalyzed by cytochrome P450 monooxygenase CYP125 and cholesterol oxidase, respectively, occur simultaneously in the strain and are followed by cascade reactions of aliphatic sidechain degradation and steroid core destruction via the known 9(10)-seco-pathway. The genes putatively related to the sterol and bile acid degradation pathways form three major clusters in the S. hirsuta genome. The sets of the genes include the orthologs of those involved in steroid catabolism in Mycobacterium tuberculosis H37Rv and Rhodococcus jostii RHA1 and related actinobacteria. Bioinformatics analysis of 52 publicly available genomes of thermophilic bacteria revealed only seven candidate strains that possess the key genes related to the 9(10)-seco pathway of steroid degradation, thus demonstrating that the ability to degrade steroids is not widespread among thermophilic bacteria.

Лаборатория микробиологической трансформации органических соединений (МТОС) была создана в 1989 г. на основе исследовательской группы в составе Отдела микробиологической трансформации органических соединений (руководитель академик Г.К. Скрябин). До 1997 г. лабораторией руководила профессор, доктор биологических наук Кира Александровна Кощеенко. Основные направления исследований лаборатории МТОС связаны с изучением микробного разнообразия стероидтрансформирующих микроорганизмов, биокаталитического потенциала микроорганизмов в отношении стероидов различной структуры, изучением особенностей катаболизма стеринов у актинобактерий, исследованием биотрансформации природных и синтетических стероидов, созданием биокаталитических систем для направленного синтеза физиологически активных стероидов на основе генетической и метаболической инженерии микробных штаммов, разработкой биопроцессов получения ценных стероидных и изопреноидных соединений и технологий полного цикла для получения фармацевтических субстанций и интермедиатов. Создана и постоянно пополняется уникальная коллекция микроорганизмов, осуществляющих структурные модификации стероидов, включая каскады реакций окислительной деградации алифатической боковой цепи, оксифункционализации стероидного ядра, введения, изомеризации и восстановления С-С-двойных связей, окисления стероидных спиртов, восстановления карбонильных групп, гидролиза стероидных эфиров, и др. На основе комплекса «-омных» исследований (геномных, транскриптомных, метаболомных) получены приоритетные данные об особенностях организации и функционирования путей катаболизма природных стеринов (фитостеринов, холестерина) у актинобактерий, выявлены наборы ключевых генов, важных для перенаправления метаболических потоков в клетке в сторону образования целевых стероидов. Созданы эффективные рекомбинантные штаммы микобактерий – клеточные биокатализаторы нового поколения, осуществляющие каскады реакций окисления боковой цепи стеринов и структурной модификации гонанового ядра стероидов с получением важных для фармацевтики стероидных и других изопреноидных соединений. Разработана методология гетерологической экспрессии эукариотических систем стероидогенеза в бактериальных хозяевах, и созданы рекомбинантные штаммы, продуцирующие в одну биотехнологическую стадию важные стероидные гормоны из природных стеринов. Разработан комплекс биотехнологий и биотехнологических методов (более 20 разработок) для производства целого ряда ценных стероидных соединений из возобновляемого растительного сырья – фитостеринов и стеринсодержащих промышленных отходов. Ряд биотехнологий масштабирован до полупромышленного уровня. Внедрение разработок может изменить облик стероидной фарминдустрии в области производства целой группы важнейших фармацевтических соединений, включая кортикостероиды, минералокортикоиды, нейростероиды, желчные кислоты. The Laboratory of Microbial Transformation of Organic Compounds (MTOC) had been created in 1989 and is having the roots from the research group at the Department of microbial transformation of organic compounds (headed by Academician George Skryabin). Professor, DSci Kira Koshcheyenko had been in charge of the Laboratory until 1997. Major research fields of the laboratory include studies of microbial diversity of steroid transforming organisms, biocatalytic properties of microorganisms towards different steroid compounds, sterol catabolism by actinobacteria, biotransformation of natural and synthetic steroids, creation of biocatalytic systems for the directed synthesis of physiologically active steroids on the base of genetic and metabolic engineering of microbial strains, bioprocess development for obtaining value-added steroid and isoprenoid compounds and full-cycle technologies for production of the active pharmaceutical ingredients and intermediates. The collection of steroid-transforming strains that performed structural modifications of steroids has been created and is continuously fulfilling. This collection includes the strains capable of performing cascades of the oxidative degradation of sterol sidechain, steroid core oxyfunctionalization, introduction, isomerization and reduction of the C-C double bonds, steroid alcohols oxidation, carbonyl groups reduction, steroid esters hydrolysis etc. Based on the results of the “-omic”s (genomics, transcriptomics, metabolomics, etc.) investigations, priority data had been obtained on the peculiarities of organization and functionality of the sterol catabolism genes in actinobacteria, key gene patterns had been revealed which are essential for re-direction of the metabolic pathways towards target steroid formation. Effective recombinant strains of mycobacteria representing cell biocatalysts of new generation have been created that perform cascade reactions of sidechain oxidation and structural modification of steroid core to produce important pharmaceutical steroids and other isoprenoids. Methodology for heterologous expression of the eukaryotic steroidogenesis system in bacterial hosts has been developed and the recombinant strains have been obtained capable of producing value-added steroid hormones in a single stage from natural sterols. Totally, over 20 biotechnologies/biotech methods for production of value-added steroids from renewable plant sterols, or sterol containing industrial wastes had been developed. Few technologies had been scaled up to the semi-industrial level. Manufacturing application will affect productions of pharmaceutical steroids including corticosteroids, mineralocorticoids, neurosteroids, bile acids etc.

Steroid microbial degradation plays a significant ecological role for biomass decomposition and removal/detoxification of steroid pollutants. In this study, the initial steps of cholesterol degradation and lithocholate bioconversion by a strain with enhanced 3-ketosteroid dehydrogenase (3-KSD) activity, Nocardioides simplex VKM Ac-2033D, were studied. Biochemical, transcriptomic, and bioinformatic approaches were used. Among the intermediates of sterol sidechain oxidation cholest-5-en-26-oic acid and 3-oxo-cholesta-1,4-dien-26-oic acid were identified as those that have not been earlier reported for N. simplex and related species. The transcriptomic approach revealed candidate genes of cholesterol and lithocholic acid (LCA) catabolism by the strain. A separate set of genes combined in cluster and additional 3-ketosteroid Δ1-dehydrogenase and 3-ketosteroid 9α-hydroxylases that might be involved in LCA catabolism were predicted. Bioinformatic calculations based on transcriptomic data showed the existence of a previously unknown transcription factor, which regulates cholate catabolism gene orthologs. The results contribute to the knowledge on diversity of steroid catabolism regulation in actinobacteria and might be used at the engineering of microbial catalysts for ecological and industrial biotechnology.

The draft genome sequence of the type strain Saccharopolyspora hirsuta subsp. hirsuta VKM Ac-666 was sequenced. This moderately thermophilic actinobacterial strain of sugarcane bagasse origin is able to transform different steroid substrates.

The dependence of biosynthesis of macrolide immunosuppressive agent tacrolimus (FK-506) in starch-containing media by the Streptomyces tsukubaensis VKM Ac-2618D strain on the molecular weight and composition of potato starch polysaccharides was studied. It was shown that the use of high-molecular branched starches as a carbon source provided a higher tacrolimus yield as compared to linear low-molecular polysaccharides at the similar culture growth indicators. The approaches providing the highest rate of tacrolimus biosynthesis by using high- and low-molecular starches and dextrins have been proposed. The biosynthesis process carrying out under the periodic batch cultivation with fractional starch feeding increased the tacrolimus yield significantly (up to 2 times), maintained biomass at a high level and reduced destructive processes rates. The maximum tacrolimus titer was observed when high-molecular potato starches were added to the culture in the retardation growth phase, while the best positive effect for low-molecular starches and dextrins additives was noted at stationary growth phase. The results could be useful in creating biotechnology for the production of pharmaceutical tacrolimus.

The 23-membered macrolide tacrolimus (FK506) is an important immunosuppressant that is widely used in the prevention of graft rejection and in the treatment of inflammatory skin diseases and immune diseases. We report here the draft genome sequence of the FK506 producer Streptomyces tsukubensis VKM Ac-2618D.

The newly synthesized (alpha/beta)-diastereomers of 6-(N-methyl-N-phenyl)aminomethylandrost-4-ene-3,17-dione (5) and 6-(N-methyl-N-phenyl)aminomethylandrost-4-en-17 beta-ol-3-one (6) were firstly investigated as substrates for the whole cells of Nocardioides simplex VKM Ac-2033D in comparison with their unsubstituted analogs, - androst-4-ene-3,17-dione (1) and androst-4-en-17 beta-ol-3-one (2).1(2)-Dehydroderivatives were identified as the major bioconversion products from all the substrates tested. When using the mixtures of (alpha/beta)-stereoisomers of 5 and 6 as the substrates, only beta-stereoisoiners of the corresponding 1,4-diene-steroids were formed. Along with 1(2) -dehydrogenation, N. simplex VKM Ac -2033D promoted oxidation of the hydroxyl group at C-17 position of 6: both 6(alpha) and 6(beta) were transformed to the corresponding 17-keto derivatives. No steroid core destruction was observed during the conversion of the 6-substituted androstanes 5 and 6, while it was significant when 1 or 2 was used as the substrate.The results suggested high potentials of N. simplex VKM Ac-2033D for the generation of novel 1(2)dehydroanalogs. (C) 2016 Elsevier Inc. All rights reserved.