Marina V. Donova

Microbial steroid transformations: current state and prospects

Studies of steroid modifications catalyzed by microbial whole cells represent a well-established research area in white biotechnology. Still, advances over the last decade in genetic and metabolic engineering, whole-cell biocatalysis in non-conventional media, and process monitoring raised research in this field to a new level. This review summarizes the data on microbial steroid conversion obtained since 2003. The key reactions of structural steroid functionalization by microorganisms are highlighted including sterol side-chain degradation, hydroxylation at various positions of the steroid core, and redox reactions. We also describe methods for enhancement of bioprocess productivity, selectivity of target reactions, and application of microbial transformations for production of valuable pharmaceutical ingredients and precursors. Challenges and prospects of whole-cell biocatalysis applications in steroid industry are discussed.

Transformation of steroids by actinobacteria: A review

Development of pharmaceutical industry is currently aimed at introducing biotechnological processes on a large-scale and thereby replacing multiple-stage chemical syntheses. Actinobacteria are efficient biocatalysts of many processes involving steroid bioconversion, which hold considerable importance for the synthesis of hormonal drugs. The potential to catalyze the conversion of a broad spectrum of steroid substrates makes it possible to expect efficient utilization of these microorganisms in development of new technologies of manufacturing steroid pharmaceutical substances. The review is a first attempt to systematize data on the potential of actinobacteria to catalyze diverse reactions of steroid transformation (such as hydroxylation, introduction and reduction of double bonds, oxidation of steroid alcohols, reduction of ketones, side chain de-esterification and degradation, etc.), with emphasis on processes of practical biotechnological importance and progress in steroid bioconversion over the last ten years.

Mycobacterium sp. mutant strain producing 9α-hydroxyandrostenedione from sitosterol

Mycobacterium sp. VKM Ac-1815D and its derivatives with altered resistance to antibacterial agents were able to produce androst-4-ene-3,17-dione (AD) as a major product from sitosterol. In this study, those strains were subjected to subsequent mutagenization by chemical agents and UV irradiation in combination with sitosterol selection pressure. The mutant Mycobacterium sp. 2-4 M was selected, being capable of producing 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) as a major product from sitosterol, with a 50% molar yield. Along with 9-OH-AD, both AD and 9α-hydroxylated metabolites with a partially degraded side-chain were formed from sitosterol by the mutant strain. The strain was unable to degrade 9-OH-AD, but degraded androsta-1,4-diene-3,17-dione (ADD), thus indicating a deficiency in steroid 1(2)-dehydrogenase and the presence of 9α-hydroxylase activity.

Evaluation of a Gluconobacter oxydans whole cell biosensor for amperometric detection of xylose

Abstract Whole cells of Gluconobacter oxydans were employed in a microbial sensor for xylose determinations using Clark-type electrodes. Bacterial cells were immobilized on chromatographic paper by simple physical adsorption and attached to the surface of the electrodes. The lower limit of xylose detection was approximately 0·5 mM and measurements were useful up to at least 20 mM xylose. Physiological buffers showed little effect on biosensor function. Responses were highly reproducible, showing a standard deviation of 6·7% over 10 consecutive measurements. Whole cell biosensors were relatively stable, retaining 60% of initial activity after 35 days of dry storage at 4°C. Xylose detection was not significantly affected by the presence of xylitol, suggesting that biosensors will be useful in monitoring conversions of these compounds. However, glucose or ethanol elicited a 10-fold higher response than xylose at equal concentrations (1 mM). Such interfering materials will need to be controlled or concurrently monitored in specific sensor applications.

FET-microbial sensor for xylose detection based on Gluconobacter oxydans cells

A potentiometric biosensor for xylose was devised utilizing Gluconobacter oxydans whole cells. Immobilization methods based on physical adsorption were used for G. oxydans cells and extracellular pH changes resulting from xylose dehydrogenation were monitored by a field effect transistor (FET). The G. oxydans, FET-based sensor detected xylose at a lower limit of 0.5 mM. From 5.0 to 30 mM xylose, the response of the sensor was linear. Expectedly, output signals were significantly suppressed by buffer (Tris-HCl). Responses were essentially stable for at least four weeks of storage and showed only a slight loss of initial xylose sensitivity. Xylitol exerted an insignificant influence on the sensors response to xylose. However, the response to glucose was 5 times higher in relation to that of xylose at the same concentration (1 mM). For xylose determinations in the presence of glucose, a two-step assay is discussed.

Comparative analysis of genes encoding key steroid core oxidation enzymes in fast-growing Mycobacterium spp. strains

A comparative genome analysis of Mycobacterium spp. VKM Ac-1815D, 1816D and 1817D strains used for efficient production of key steroid intermediates (androst-4-ene-3,17-dione, AD, androsta-1,4-diene-3,17-dione, ADD, 9α-hydroxy androst-4-ene-3,17-dione, 9-OH-AD) from phytosterol has been carried out by deep sequencing. The assembled contig sequences were analyzed for the presence putative genes of steroid catabolism pathways. Since 3-ketosteroid-9α-hydroxylases (KSH) and 3-ketosteroid-Δ(1)-dehydrogenase (Δ(1) KSTD) play key role in steroid core oxidation, special attention was paid to the genes encoding these enzymes. At least three genes of Δ(1) KSTD (kstD), five genes of KSH subunit A (kshA), and one gene of KSH subunit B of 3-ketosteroid-9α-hydroxylases (kshB) have been found in Mycobacterium sp. VKM Ac-1817D. Strains of Mycobacterium spp. VKM Ac-1815D and 1816D were found to possess at least one kstD, one kshB and two kshA genes. The assembled genome sequence of Mycobacterium sp. VKM Ac-1817D differs from those of 1815D and 1816D strains, whereas these last two are nearly identical, differing by 13 single nucleotide substitutions (SNPs). One of these SNPs is located in the coding region of a kstD gene and corresponds to an amino acid substitution Lys (135) in 1816D for Ser (135) in 1815D. The findings may be useful for targeted genetic engineering of the biocatalysts for biotechnological application.

Dissolution of a Mixture of Steroids in Cyclodextrin Solutions: a Model Description

Cyclodextrins (CDs) are widely used in pharmacology as biocompatible and nontoxic carriers of biologically active agents. The use of various drugs in the form of CD-based inclusion complexes (of the host – guest type) increases the solubility and stability of the parent drugs, facilitates delivery of the hydrophobic components to the target organ, and reduces undesired side effects [1]. The ideal partners for CD-based inclusion complexes are steroidal structures [2]. However, the use of several steroids forming a common complex with CD [3] in pharmaceutical preparations poses a question of the possible mutual influence and the resulting solubility under the conditions of competitive binding. The purpose of this study was to establish the laws of dissolution for a mixture of steroids in CD solutions. For this purpose, we have developed a simple but effective model of solubility for such steroid mixtures.

Screening of mycelial fungi for 7α- and 7β-hydroxylase activity towards dehydroepiandrosterone

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.

Extracellular 3β-hydroxysteroid oxidase of Mycobacterium vaccae VKM Ac-1815D

Extracellular 3beta-hydroxysteroid oxidase (SO) has been isolated from cell-free cultivation broth at the growth of Mycobacterium vaccae VKM Ac-1815D on glycerol-mineral medium in the presence of sitosterol. The enzyme is responsible for the transformation of 3beta-hydroxy-5-ene- to 3-keto-4-ene-moiety of steroids including dehydrogenation of 3beta-hydroxy function followed by delta5-->delta4 isomerization. 6-Hydroxy-4-sitosten-3-one and 6-hydroxy-4-androsten-3,17-dione were revealed among the metabolites at the incubation of the enzyme preparations with sitosterol and dehydroepiandrosterone (DHEA), respectively. The enzyme was strongly NADH or NADPH dependent. SO has been purified over 300-fold using cultivation broth concentration on hollow fibers followed by fractionation by ammonium sulphate, column chromatography on DEAE-Toyopearl, hydroxyapatite Bio-Gel HTP and double gel-filtration on Bio-Gel A 0.5 M. SDS-electrophoresis gave a molecular mass estimate of 62 +/- 4 kDa. The purified SO obeyed Michaelis-Menten kinetics, double reciprocal plots kinetics revealed Km value towards DHEA 5 x 10(-4) M. Along with SO activity, 17-hydroxysteroid dehydrogenase (17-OH SDH) and 3-ketosteroid-1(2)-dehydrogenase (1(2)-SDH) activities were detected in cell-free cultivation broth. The extracellular steroid transforming activities of C-17-ketosteroid producing mycobacteria were hitherto unreported.

Cholesterol oxidase ChoD is not a critical enzyme accounting for oxidation of sterols to 3-keto-4-ene steroids in fast-growing Mycobacterium sp. VKM Ac-1815D

Fast-growing strain of Mycobacterium sp. VKM Ac-1815D is capable of effective oxidizing of sterols (phytosterol, cholesterol, ergosterol) to androstenedione and other valuable 3-oxo-steroids. To elucidate the role of cholesterol oxidase in sterol catabolism by the strain, the choD gene has been cloned and sequenced. The deduced gene product (M(r) 63.5kDa) showed homologies over its entire length to a large number of proteins belonging to the InterPro-family EPR006076, which includes various FAD dependent oxidoreductases. The expression of choD in Escherichia coli was shown to result in the synthesis of membrane associated cholesterol oxidase. In addition to cholesterol, the enzyme oxidized β-sitosterol, dehydroepiandrosterone, ergosterol, pregnenolone, and lithocholic acid. Knock-out of choD in Mycobacterium sp. VKM Ac-1815D strain was obtained by the gene replacement technique. The mutant strain transformed sitosterol forming exclusively 3-keto-4-ene steroids with androstenedione as a major product, thus evidencing that choD knock out did not abrogate sterol A-ring oxidation. The results indicated that ChoD is not a critical enzyme responsible for modification of 3β-hydroxy-5-ene- to 3-keto-4-ene steroids in Mycobacterium sp. VKM Ac-1815D. Article from a special issue on steroids and microorganisms.