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.

Draft genome sequence of soil isolate Mycolicibacterium fortuitum DVD-1301.

Some strains of Mycolicibacterium 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 Mycolicibacterium fortuitum DVD-1301 isolated in the floodplain of the river Oka. The genome contains a full set of steroid catabolic genes.

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 C19 or C22 skeleton. Here we describe a method for site-directed mutagenesis of Mycolicibacterium neoaurum strains, bioconversion from phytosterol, and selective purification of C23 steroid 24-norchol-4-ene-3,22-dione (24-NCED) and C22 steroid 20-hydroxymethylpregn-4-ene-3-one (20-HMP). The yields of crystalline products with 95% purity by the method here described are 2.74 ± 0.085 g for 24-NCED and 1.42 ± 0.085 g for 20-HMP from 10 g/L phytosterol. 20-HMP is recognized as the key precursor in chemical syntheses of pharmaceutical corticosteroids and 24-NCED is a promising synthon for the synthesis of valuable steroids and own potent biological activity.

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

Mycolicibacterium smegmatis 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.

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. However, the set of the genes with products that are involved in phytosterol oxidation, their organisation and regulation remain poorly understood. RESULTS: High-throughput sequencing of the global transcriptomes of the Mycobacterium sp. VKM Ac-1817D cultures grown with or without phytosterol was carried out. In the presence of phytosterol, the expression of 260 genes including those related to steroid catabolism pathways significantly increased. Two of the five genes encoding the oxygenase unit of 3-ketosteroid-9α-hydroxylase (kshA) were highly up-regulated in response to phytosterol (55- and 25-fold, respectively) as well as one of the two genes encoding its reductase subunit (kshB) (40-fold). Only one of the five putative genes encoding 3-ketosteroid-∆1-dehydrogenase (KstD_1) was up-regulated in the presence of phytosterol (61-fold), but several substitutions in the conservative positions of its product were revealed. Among the genes over-expressed in the presence of phytosterol, several dozen genes did not possess binding sites for the known regulatory factors of steroid catabolism. In the promoter regions of these genes, a regularly occurring palindromic motif was revealed. The orthologue of TetR-family transcription regulator gene Rv0767c of M. tuberculosis was identified in Mycobacterium sp. VKM Ac-1817D as G155_05115. CONCLUSIONS: High expression levels of the genes related to the sterol side chain degradation and steroid 9α-hydroxylation in combination with possible defects in KstD_1 may contribute to effective 9α-hydroxyandrost-4-ene-3,17-dione accumulation from phytosterol provided by this biotechnologically relevant strain. The TetR-family transcription regulator gene G155_05115 presumably associated with the regulation of steroid catabolism. The results are of significance for the improvement of biocatalytic features of the microbial strains for the steroid industry.

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

Modified ß-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. Phytosterol bioconversion with 9α-hydroxy-androst-4-ene-3,17-dione accumulation by Mycobacterium sp. VKM Ac-1817D in the presence of methylated ß-cyclodextrin (MCD) was investigated. RNA sequencing of the whole transcriptomes in different combinations of phytosterol and MCD showed a similar expression level of the steroid catabolism genes related to the KstR-regulon and was responsible for side chain and initial steps of steroid core oxidation; whereas, induction levels of the genes related to the KstR2-regulon were attenuated in the presence of MCD in this strain. The data were attenuated with quantitative real-time PCR.The results contribute to the understanding of cyclodextrin effects on microbial steroid conversion and provide a basis for the use of cyclodextrins as expression enhancers for studies of sterol catabolism in actinobacteria.

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. The genome consists of a single circular 6,324,222-bp chromosome with a G+C content of 66.2% and encodes approximately 6,000 CDSs, 54 tRNAs, and 6 rRNAs.

Complete Genome Sequence of Sterol-Transforming Mycobacterium neoaurum Strain VKM Ac-1815D.

Mycobacterium neoaurum strain VKM Ac-1815D produces 4-androstene-3,17-dione as a major compound from phytosterols. Here, we report the complete genome sequence of the strain. The genome consists of a single circular 5,438,190-bp chromosome, with a G+C content of 66.88%, containing 5,318 putative open reading frames (ORFs), 46 tRNAs, and 6 rRNAs. Arrays of cholesterol metabolism genes are randomly clustered throughout the chromosome.

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 ß-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 3ß-hydroxy-5-ene- to 3-keto-4-ene steroids in Mycobacterium sp. VKM Ac-1815D. Article from a special issue on steroids and microorganisms.

Microbial side-chain degradation of ergosterol and its 3-substituted derivatives: a new route for obtaining of deltanoids.

The strain of Mycobacterium sp. VKM Ac-1815D was found to convert ergosterol and its 3-acetate mainly to androst-4-ene-3,17-dione (AD) thus demonstrating ability to reduce 7(8)-double bond and hydrolyze sterol ester in addition to oxidation of 3beta-hydroxy group, Delta(5)-Delta(4) isomerization and side-chain degradation. Ergosterol bioconversion in the presence of isoflavones and ions of some bivalent metals - known inhibitors of 3beta-hydroxysteroid dehydrogenase, did not alter products composition. Protection of ergosterol 3beta-hydroxyl with methoxymethyl group allowed the formation of bioconversion products retaining the Delta(5,7)-configuration. The major product was identified by mass-spectrometry and proton NMR as 3-methoxymethoxy-androsta-5,7-diene-17-one (MA). The MA producing activity was found to be inducible with sterols, cholestenone or lithocholic acid, but not with dehydroepiandrosterone, AD, androsta-1,4-ene-3,17-dione or organic acids. Under the optimized conditions, the yield of MA reached 5g/l from 10g/l O-methoxymethyl-ergosterol (approx. 60% molar conversion) for 120h. The results might be applied at the production of novel vitamin D derivatives.