Boldenone and Testosterone Production from Phytosterol via One-Pot Cascade Biotransformations.

Testosterone (TS) and its 1(2)-dehydrogenated derivative boldenone (BD) are widely used in medicine, veterinary science and as precursors in organic synthesis of many therapeutic steroids. Green production of these compounds is possible from androstenedione (AD) enzymatically, or from phytosterol (PS) using fermentation stages. In this study, the ascomycete sp.

Reconstruction of the Steroid 1(2)-Dehydrogenation System from VKM Ac-2033D in 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 and 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 () from VKM Ac-2033D.

Draft genome sequence of soil isolate DVD-1301.

Some strains of possess high sterol-oxidizing activity and are used in the pharmaceutical industry for the production of steroid precursors. Herein, we report a draft genome sequence of the soil-dwelling DVD-1301 isolated in the floodplain of the river Oka. The genome contains a full set of steroid catabolic genes.

transcriptome assembly of sp. VKM F-3040, a promising steroid-modifying ascomycete.

This research presents transcriptome shotgun assembly for sp. VKM F-3040, which is a putative fungal strain able to modify androstane steroids with production of 7-hydroxy and 17-hydroxylated derivatives-key intermediates in the synthesis of pharmaceutical ingredients. The data are of importance for creating novel microbial biocatalysts.

Obtaining of 24-Norchol-4-ene-3,22-dione from Phytosterol with Mutants of Mycolicibacterium neoaurum.

Engineered mutants of Mycolicibacterium spp. are known producers of valuable steroid synthons with C or C skeleton. Here we describe a method for site-directed mutagenesis of Mycolicibacterium neoaurum strains, bioconversion from phytosterol, and selective purification of C steroid 24-norchol-4-ene-3,22-dione (24-NCED) and C steroid 20-hydroxymethylpregn-4-ene-3-one (20-HMP).

Selective Microbial Conversion of DHEA into 7α-OH-DHEA.

7α-Hydroxy dehydroepiandrosterone (7α-OH-prasterone, 7α-OH-DHEA) is a key steroid intermediate in the synthesis of valuable pharmaceuticals widely used in the treatment of autoimmune illness, rheumatoid arthritis, colitis, and other severe diseases. The steroid can be produced using a filamentous fungus, which is capable of regio- and stereospecific hydroxylation of the steroid 3β-alcohol (DHEA) in the allylic position C7. Here, we describe a method for highly selective microbial production of 7α-OH-DHEA from DHEA using the zygomycete Backusella lamprospora VKM F-944.

Cholesterol Assay Based on Recombinant Cholesterol Oxidase, ABTS, and Horseradish Peroxidase.

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.

Current Trends and Perspectives in Microbial Bioconversions of Steroids.

The microbiological transformation of sterols is currently the technological basis for the industrial production of valuable steroid precursors, the so-called synthons, from which a wide range of steroid and indane isoprenoids are obtained by combined chemical and enzymatic routes. These compounds include value-added corticoids, neurosteroids, sex hormones, bile acids, and other terpenoid lipids required by the medicine, pharmaceutical, food, veterinary, and agricultural industries.Progress in understanding the molecular mechanisms of microbial degradation of steroids, and the development and implementation of genetic technologies, opened a new era in steroid biotechnology.

Draft Genome Sequence of Mycolicibacterium smegmatis VKM Ac-1171 Contains Full Set of Sterol Catabolic Genes.

VKM Ac-1171 is a saprotrophic bacterium that was isolated several decades ago and is deposited in microbial collections around the world. We report here a draft genome sequence of the strain. Annotation of the genome revealed the presence of a complete set of genes related to the sterol catabolic pathway.

Bioproduction of testosterone from phytosterol by Mycolicibacterium neoaurum strains: "one-pot", two modes.

The main male hormone, testosterone is obtained from cheap and readily available phytosterol using the strains of Mycolicibacterium neoaurum VKM Ac-1815D, or Ac-1816D. During the first "oxidative" stage, phytosterol (5-10 g/L) was aerobically converted by Ac-1815D, or Ac-1816D to form 17-ketoandrostanes: androstenedione, or androstadienedione, respectively. At the same bioreactor, the 17-ketoandrostanes were further transformed to testosterone due to the presence of 17β-hydroxysteroid dehydrogenase activity in the strains ("reductive" mode).

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.

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

Background: Bacterial 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 Δ-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.

Genome-Wide Transcriptome Profiling Provides Insight on Cholesterol and Lithocholate Degradation Mechanisms in 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, VKM Ac-2033D, were studied. Biochemical, transcriptomic, and bioinformatic approaches were used.

An Efficient Procedure for the Synthesis of 21-Acetoxypregna-1,4,9(11),16- tetraene-3,20-dione.

Background: Halogenated corticosteroids are widely used in medicine, and the global need of these steroidal APIs is estimated to be 40 - 70 tons, annually. Vietnam currently imports the pharmaceutical compounds up to 90%, in particular 100% of steroidal drugs. Currently, industrial production is based on the chemical syntheses of corticosteroids from either 16- dehydropregnenolone acetate (obtained from diosgenin) or androstenedione (obtained from phytosterol).

Draft Genome Sequence of the Moderately Thermophilic Actinobacterial Steroid-Transforming Saccharopolyspora hirsuta subsp. Strain VKM Ac-666.

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

Genome-wide response on phytosterol in 9-hydroxyandrostenedione-producing strain of Mycobacterium sp. VKM Ac-1817D.

Background: Aerobic side chain degradation of phytosterols by actinobacteria is the basis for the industrial production of androstane steroids which are the starting materials for the synthesis of steroid hormones. A native strain of Mycobacterium sp. VKM Ac-1817D effectively produces 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) from phytosterol, but also is capable of slow steroid core degradation.

Draft Genome Sequence of FK506-Producing Strain VKM Ac-2618D.

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 VKM Ac-2618D.

Obtaining of 11α-Hydroxyandrost-4-ene-3,17-dione from Natural Sterols.

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.

Genome Sequencing of Steroid-Producing Bacteria with Illumina Technology.

Illumina technology is widely used for bacterial whole-genome sequencing due to its simplicity, cheapness, reliability, and abundant software for manipulation with raw data. Illumina technology belongs to a second generation of whole genome sequencing that yields great amount of short reads for genome regions. Genomic DNA is fragmented to short pieces.

Steroid Bioconversions.

Steroid modifications by selected wild-type and engineered strains of microorganisms became an effective tool for the production of high-valued steroidal drugs and their precursors for the pharmaceutical industry. Some microorganisms are effective at the performance of sterol side-chain degradation, oxyfunctionalization of steroid core, and redox reactions at different positions of the steroid molecule. A number of bioprocesses using steroid-transforming microbial strains are well established on an industrial level.

Effect of methyl-β-cyclodextrin on gene expression in microbial conversion of phytosterol.

Modified β-cyclodextrins are widely used for the enhancement of microbial conversions of lipophilic compounds such as steroids. Multiple mechanisms of cyclodextrin-mediated enhancement of phytosterol bioconversion by mycobacteria had previously been shown to include steroid solubilization, alterations in the cell wall permeability for both steroids and nutrients, facilitation of protein leaking, and activity suppression of some steroid-transforming enzymes.In this work, we studied whether cyclodextrins might affect expression of the genes involved in the steroid catabolic pathway.

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.

Complete Genome Sequence of Mycobacterium sp. Strain VKM Ac-1817D, Capable of Producing 9α-Hydroxy-androst-4-ene-3,17-dione from Phytosterol.

Mycobacterium sp. strain VKM Ac-1817D is capable of converting phytosterol into 9α-hydroxy androst-4-ene-3,17-dione (9-OH-AD), which is a valuable intermediate for the steroid pharmaceutical industry. Here, a complete genome sequence of the strain is reported.