Simplex VKM Ac-2033D Research Articles

Reconstruction of the Steroid 1(2)-Dehydrogenation System from Nocardioides simplex VKM Ac-2033D in Mycolicibacterium Hosts.

Microbial 1(2)-dehydrogenation of 3-ketosteroids is an important basis for the production of many steroid pharmaceuticals and synthons. When using the wild-type strains for whole cell catalysis, the undesirable reduction of the 20-carbonyl group, or 1(2)-hydrogenation, was observed. In this work, the recombinant strains of Mycolicibacterium neoaurum and Mycolicibacterium smegmatis were constructed with blocked endogenous activity of 3-ketosteroid-9α-hydroxylase, 3-ketosteroid-1(2)-dehydrogenase (3-KSD), and expressing 3-KSD encoded by the gene KR76_27125 (kstD2NS) from Nocardioides simplex VKM Ac-2033D. The in vivo activity of the obtained recombinant strains against phytosterol, 6α-methyl-hydrocortisone, and hydrocortisone was studied. When using M. smegmatis as the host strain, the 1(2)-dehydrogenation activity of the constructed recombinant cells towards hydrocortisone was noticeably higher compared to those on the platform of M. neoaurum. A comparison of the strengths of inducible acetamidase and constitutive hsp60 promoters in M. smegmatis provided comparable results. Hydrocortisone biotransformation by M. smegmatis BD/pMhsp_k expressing kstD2NS resulted in 95.4% prednisolone yield, and the selectivity preferred that for N. simplex. Mycolicibacteria showed increased hydrocortisone degradation at 35 °C compared to 30 °C. The presence of endogenous steroid catabolism in Mycolicibacterium hosts does not seem to confer an advantage for the functioning of KstD2NS. The results allow for the evaluation of the prospects for the development of simple technological methods for the selective 1(2)-dehydrogenation of 3-ketosteroids by growing bacterial cells.

Recombinant Extracellular Cholesterol Oxidase from Nocardioides simplex.

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.

Different genome-wide transcriptome responses of Nocardioides simplex VKM Ac-2033D to phytosterol and cortisone 21-acetate

BackgroundBacterial degradation/transformation of steroids is widely investigated to create biotechnologically relevant strains for industrial application. The strain of Nocardioides simplex VKM Ac-2033D is well known mainly for its superior 3-ketosteroid Δ1-dehydrogenase activity towards various 3-oxosteroids and other important reactions of sterol degradation. However, its biocatalytic capacities and the molecular fundamentals of its activity towards natural sterols and synthetic steroids were not fully understood. In this study, a comparative investigation of the genome-wide transcriptome profiling of the N. simplex VKM Ac-2033D grown on phytosterol, or in the presence of cortisone 21-acetate was performed with RNA-seq.ResultsAlthough the gene patterns induced by phytosterol generally resemble the gene sets involved in phytosterol degradation pathways in mycolic acid rich actinobacteria such as Mycolicibacterium, Mycobacterium and Rhodococcus species, the differences in gene organization and previously unreported genes with high expression level were revealed. Transcription of the genes related to KstR- and KstR2-regulons was mainly enhanced in response to phytosterol, and the role in steroid catabolism is predicted for some dozens of the genes in N. simplex. New transcription factors binding motifs and new candidate transcription regulators of steroid catabolism were predicted in N. simplex.Unlike phytosterol, cortisone 21-acetate does not provide induction of the genes with predicted KstR and KstR2 sites. Superior 3-ketosteroid-Δ1-dehydrogenase activity of N. simplex VKM Ac-2033D is due to the kstDs redundancy in the genome, with the highest expression level of the gene KR76_27125 orthologous to kstD2, in response to cortisone 21-acetate. The substrate spectrum of N. simplex 3-ketosteroid-Δ1-dehydrogenase was expanded in this study with progesterone and its 17α-hydroxylated and 11α,17α-dihydroxylated derivatives, that effectively were 1(2)-dehydrogenated in vivo by the whole cells of the N. simplex VKM Ac-2033D.ConclusionThe results contribute to the knowledge of biocatalytic features and diversity of steroid modification capabilities of actinobacteria, defining targets for further bioengineering manipulations with the purpose of expansion of their biotechnological applications.

Genome-Wide Transcriptome Profiling Provides Insight on Cholesterol and Lithocholate Degradation Mechanisms in Nocardioides simplex VKM Ac-2033D.

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.

Biosynthesis of desferrioxamine siderophores initiated by decarboxylases: A functional investigation of two lysine/ornithine-decarboxylases from Gordonia rubripertincta CWB2 and Pimelobacter simplex 3E

Lysine is a precursor for desferrioxamine siderophore biosynthesis. The pathway is often initiated by lysine decarboxylases. However, little is known about those enzymes from Actinobacteria which represents a diverse class of desferrioxamine producers. In this study we focused on the genes grdesA form Gordonia rubripertincta CWB2 and psdesA from Pimelobacter simplex VkMAC-2033D that encode decarboxylases presumed to be involved in the synthesis of desferrioxamine siderophores. The corresponding proteins GrDesA and PsDesA, were heterologously produced in Escherichia coli and purified. PsDesA was isolated bound to the cofactor pyridoxal 5-phosphate and GrDesA was purified in its apo form. PsDesA showed a moderate substrate preference for lysine (Km = 0.17 mM, kcat = 0.26 s−1) compared to ornithine (Km = 0.13 mM, kcat = 0.14 s−1), while GrDesA exhibited specificity for lysine (Km = 0.13 mM, kcat = 1.2 s−1) compared to ornithine (Km = 2.9 mM, kcat = 0.18 s−1). The maximum decarboxylase activity of PsDesA was achieved at pH 7.5 at 35 °C, although PsDesA was stable up to 40°, its relative activity decreased significantly at 50 °C. The temperature optimum (40 °C) and thermostability of GrDesA were likewise, but it exhibited maximum activity at pH range 8.0–8.5, and sharply decreased outside of this range. The expression and characterization of these two decarboxylases provides insight into the biosynthetic pathway of desferrioxamines from G.rubripertincta and P. simplex and supports the functional annotation of related pathways.

Microbiological synthesis of stereoisomeric 7(α/β)-hydroxytestololactones and 7(α/β)-hydroxytestolactones.

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 1H-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.

Bioconversion of 6-(N-methyl-N-phenyl)aminomethyl androstane steroids by Nocardioides simplex

The newly synthesized (α/β)-diastereomers of 6-(N-methyl-N-phenyl)aminomethylandrost-4-ene-3,17-dione (5) and 6-(N-methyl-N-phenyl)aminomethylandrost-4-en-17β-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-17β-ol-3-one (2).1(2)-Dehydroderivatives were identified as the major bioconversion products from all the substrates tested. When using the mixtures of (α/β)-stereoisomers of 5 and 6 as the substrates, only β-stereoisomers 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(α) and 6(β) 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.

Genome-wide bioinformatics analysis of steroid metabolism-associated genes in Nocardioides simplex VKM Ac-2033D

Actinobacteria comprise diverse groups of bacteria capable of full degradation, or modification of different steroid compounds. Steroid catabolism has been characterized best for the representatives of suborder Corynebacterineae, such as Mycobacteria, Rhodococcus and Gordonia, with high content of mycolic acids in the cell envelope, while it is poorly understood for other steroid-transforming actinobacteria, such as representatives of Nocardioides genus belonging to suborder Propionibacterineae. Nocardioides simplex VKM Ac-2033D is an important biotechnological strain which is known for its ability to introduce ∆(1)-double bond in various 1(2)-saturated 3-ketosteroids, and perform convertion of 3β-hydroxy-5-ene steroids to 3-oxo-4-ene steroids, hydrolysis of acetylated steroids, reduction of carbonyl groups at C-17 and C-20 of androstanes and pregnanes, respectively. The strain is also capable of utilizing cholesterol and phytosterol as carbon and energy sources. In this study, a comprehensive bioinformatics genome-wide screening was carried out to predict genes related to steroid metabolism in this organism, their clustering and possible regulation. The predicted operon structure and number of candidate gene copies paralogs have been estimated. Binding sites of steroid catabolism regulators KstR and KstR2 specified for N. simplex VKM Ac-2033D have been calculated de novo. Most of the candidate genes grouped within three main clusters, one of the predicted clusters having no analogs in other actinobacteria studied so far. The results offer a base for further functional studies, expand the understanding of steroid catabolism by actinobacteria, and will contribute to modifying of metabolic pathways in order to generate effective biocatalysts capable of producing valuable bioactive steroids.

Complete Genome Sequence of Steroid-Transforming Nocardioides simplex VKM Ac-2033D.

Nocardioides simplex VKM Ac-2033D is an effective microbial catalyst for 3-ketosteroid 1(2)-dehydrogenation, and it is capable of effective reduction of carbonyl groups at C-17 and C-20, hydrolysis of acetylated steroids, and utilization of natural sterols. Here, the complete genome sequence is reported. An array of genes related to steroid metabolic pathways have been identified.

21-Acetoxy-pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione conversion by Nocardioides simplex VKM Ac-2033D

The conversion of 21-acetoxy-pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione ( I) by Nocardioides simplex VKM Ac-2033D was studied purposed selective production of its 1(2)-dehydroanalogues—value precursors in the synthesis of modern glucocorticoids starting from 9α-hydroxyandrostenes. 21-Acetoxy-pregna-1(2),4(5),9(11),16(17)-tetraene-21-ol-3,20-dione ( II), pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione ( III) and pregna-1(2),4(5),9(11),16(17)-tetraene-21-ol-3,20-dione ( IV) were revealed as metabolites, and the structures were confirmed by mass spectrometry and 1 H nuclear magnetic resonance (NMR) spectroscopy. The metabolic pathways of I by N. simplex included 1(2)-dehydrogenation and deacetylation. The sequence of the reactions was shown to depend on the transformation conditions. The presence of both soluble and membrane associated steroid esterases in N. simplex was demonstrated using cell fractionation. Unlike inducible 1(2)-dehydrogenase, steroid esterase was shown to be constitutive. The conditions providing selective accumulation of II from I by whole N. simplex cells were determined.

Microbial conversion of pregna-4,9(11)-diene-17α,21-diol-3,20-dione acetates by Nocardioides simplex VKM Ac-2033D

The conversion of pregna-4,9(11)-diene-17α,21-diol-3,20-dione 21-acetate ( I) and 17,21-diacetate ( VI) by Nocardioides simplex VKM Ac-2033D was studied. The major metabolites formed from I were identified as pregna-1,4,9(11)-triene-17α,21-diol-3,20-dione 21-acetate ( II) and pregna-1,4,9(11)-triene-17α,21-diol-3,20-dione ( IV). Pregna-4,9(11)-diene-17α,21-diol-3,20-dione ( III) and pregna-1,4,9(11)-triene-17α,20β,21-triol-3-one ( V) were formed in minorities. Biotransformation products formed from VI were pregna-1,4,9(11)-triene-17α,21-diol-3,20-dione 17,21-diacetate ( VII), pregna-1,4,9(11)-triene-17α,21-diol-3,20-dione 21-acetate ( II), pregna-1,4,9(11)-triene-17α,21-diol-3,20-dione ( IV), pregna-1,4,9(11)-triene-17α,21-diol-3,20-dione 17-acetate ( VIII), pregna-1,4,9(11)-triene-17α,20β,21-triol-3-one ( V). The conversion pathways were proposed including 1(2)-dehydrogenation, deacetylation, 20β-reduction and non-enzymatic migration of acyl group from position 17 to 21. The conditions providing predominant accumulation of pregna-1,4,9(11)-triene-17α,21-diol-3,20-dione 21-acetate ( II) from I and pregna-1,4,9(11)-triene-17α,21-diol-3,20-dione 17-acetate ( VIII) from VI in a short-term biotransformation were determined.

The 1(2)-dehydrogenation of steroid substrates by Nocardioides simplex VKM Ac-2033D

The bacterium formerly known as Arthrobacter globiformis 193 has high 1(2)-dehydrogenase activity toward pharmaceutically important steroids, 9(11)-dehydrocortexolone in particular. The complex analysis of the morphostructural, physiological, biochemical, and phylogenetic properties of this bacterium allowed us to reclassify it into Nocardioides simplex (N. simplex VKM Ac-2033D).