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.

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.

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.

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.

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.

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.

The capability of 54 selected actinobacteria strains of different phyla to convert deoxycholic (DCA) and cholic (CA) acids under aerobic conditions was studied. Except for the two species, the strains did not grow on DCA (1g/l) as a sole carbon source, but some of them effectively converted DCA performing 7β- and 9α- hydroxylation, 3α- and 12α-dehydrogenation, partial cleavage of the isoprenoic side chain and Δ4-dehydrogenation. Ursocholic acid, 9α-hydroxy-3,12-dioxo-23,24-bisnorchol-4-ene-22-oic acid, 3-keto-DCA and other end metabolites had been firstly identified in the actinobacteria strains. The total yield of 12α-hydroxy-3-oxo-chol-4-ene-24-oic and 3,12-dioxochol-4-ene-24-oic acids from DCA with Rhodococcus erythropolis VKM Ac-1152 reached 95%. Almost 80% DCA were converted to 9α-hydroxy-3,12-dioxo-23,24-bisnorchol-4-en-22-oic acid by Rhodococcus sp. MTS-77. Unlike DCA, cholic acid (CA) was confirmed to be a growth substrate for majority of the examined strains, but only three Rhodococcus strains exhibited 7α- and/or 12α-HSDH activities thus forming 7-keto-DCA and 12-keto-chenodeoxycholic acid as major products from CA. Steroid metabolites were identified by TLC, GC, MS, 1H- and 13C NMR analyses.The results may contribute to the knowledge of biocatalytic potential of diverse soil-dwelling actinobacteria towards bile acids, and could be applied at the development of novel bioprocesses for production of the valuable cholanic acids.

More than 100 filamentous fungi strains, mostly ascomycetes and zygomycetes from different phyla, were screened for the ability to convert deoxycholic acid (DCA) to valuable bile acid derivatives. Along with 11 molds which fully degraded DCA, several strains were revealed capable of producing cholic acid, ursocholic acid, 12-keto-lithocholic acid (12-keto-LCA), 3-keto-DCA, 15β-hydroxy-DCA and 15β-hydroxy-12-oxo-LCA as major products from DCA. The last metabolite was found to be a new compound. The ability to catalyze the introduction of a hydroxyl group at the 7(α/β)-positions of the DCA molecule was shown for 32 strains with the highest 7β-hydroxylase activity level for Fusarium merismoides VKM F-2310. Curvularia lunata VKM F-644 exhibited 12α-hydroxysteroid dehydrogenase activity and formed 12-keto-LCA from DCA. Acremonium rutilum VKM F-2853 and Neurospora crassa VKM F-875 produced 15β-hydroxy-DCA and 15β-hydroxy-12-oxo-LCA, respectively, as major products from DCA, as confirmed by MS and NMR analyses. For most of the positive strains, the described DCA-transforming activity was unreported to date. The presented results expand the knowledge on bile acid metabolism by filamentous fungi, and might be suitable for preparative-scale exploitation aimed at the production of marketed bile acids.

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.

Selected actinobacteria and filamentous fungi of different taxonomy were screened for the ability to carry out regio- and stereospecific hydroxylation of lithocholic acid (LCA) at position 7β. The production of ursodeoxycholic acid (UDCA) was for the first time shown for the fungal strains of Bipolaris, Gibberella, Cunninghamella and Curvularia, as well as for isolated actinobacterial strains of Pseudonocardia, Saccharothrix, Amycolatopsis, Lentzea, Saccharopolyspora and Nocardia genera. Along with UDCA, chenodeoxycholic (CDCA), deoxycholic (DCA), cholic (CA), 7-ketodeoxycholic and 3-ketodeoxycholic acids were detected amongst the metabolites by some strains. A strain of Gibberella zeae VKM F-2600 expressed high level of 7β-hydroxylating activity towards LCA. Under optimized conditions, the yield of UDCA reached 90% at 1 g/L of LCA and up to 60% at a 8-fold increased substrate loading. The accumulation of the major by-product, 3-keto UDCA, was limited by using selected biotransformation media.

The ability of 485 fungal strains is studied for catalysis of the process of 7α, 15α-dihydroxylation of dehydroepiandrosterone (DHEA, 3β-hydroxy-5-androstene-17-one), a key intermediate of the synthesis of physiologically active compounds. The ability for the formation of 3β, 7α, 15α-trihydroxy-5-androstene-17-one ( 7α, 15α-diOH-DHEA ) was found for the first time for representatives of 12 genera, eight families, and six orders of ascomycetes, eight genera, four families, and one order of zygomycetes, one genus, one family, and one order of basidiomycetes, and four genera of mitosporic fungi. The most active strains are found among genera Acremonium, Gibberella, Fusarium , and Nigrospora . In the process of transformation of DHEA (2 g/l) by strains of Fusarium oxysporum VKM F-1600 and Gibberella zeae BKM F-2600, the molar yield was 63 and 68%, respectively. Application of the revealed active strains of microorganisms opens prospects for the efficient production of key intermediates of synthesis of modern medical preparations.

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.

In total, 481 fungal strains were screened for the ability to carry out 7(α/β)-hydroxylation of dehydroepiandrosterone (DHEA, 3β-hydroxy-5-androsten-17-one). Representatives of 31 genera of 15 families and nine orders of ascomycetes, 17 genera of nine families and two orders of zygomycetes, two genera of two families and two orders of basidiomycetes, and 14 genera of mitosporic fungi expressed 7(α/β)-hydroxylase activity. The majority of strains were able to introduce a hydroxyl group to position 7α. Active strains selectively producing 3β,7α-dihydroxy-5-androsten-17-one were found among Actinomucor, Backusella, Benjaminiella, Epicoccum, Fusarium, Phycomyces and Trichothecium, with the highest yield of 1.25 and 1.9 g L−1 from 2 and 5 g L−1 DHEA, respectively, reached with F. oxysporum. Representatives of Acremonium, Bipolaris, Conidiobolus and Curvularia formed 3β,7β-dihydroxy-5-androsten-17-one as a major product from DHEA. The structures of the major steroid products were confirmed by TLC, gas chromatography (GC), mass spectra (MS), and 1H-NMR analyses.

Carbazole was metabolized by Aspergillus flavus VKM F-1024 forming few monohydroxylated products. The structure of metabolites was determined by TLC, GC, MS and (1)H NMR analyses. 3-Hydroxycarbazole was revealed as a major bioconversion product, 1-hydroxy- and 2-hydroxycarbazoles were observed as minor products. In the presence of 1-benzoylindole, the hydroxylation position shifted toward preferable accumulation of 2-hydroxycarbazole and the formation of 2,6- and 2,7-dihydroxycarbazoles. This effect and microbial formation of these metabolites have never been reported before. At the conversion of N-acetyl- and N-benzoylcarbazoles, carbazole was the major product, while 1-, 2- and 3-monohydroxycarbazoles were formed in small amounts.