Circulating adrenal and gonadal steroid hormones heterogeneity in active young males and the contribution of 11-oxy androgens.

The classical androgens, testosterone and dihydrotestosterone, together with dehydroepiandrosterone, the precusrsor to all androgens, are generally included in diagnostic steroid evaluations of androgen excess and deficiency disorders and monitored in androgen replacement and androgen suppressive therapies. The C11-oxy androgens also contribute to androgen excess disorders and are still often excluded from clinical and research-based steroids analysis. The contribution of the C11-oxy androgens to the androgen pool has not been considered in androgen deficiency. An exploratory investigation into circulating adrenal and gonadal steroid hormones in men was undertaken as neither the classical androgens nor the C11-oxy androgens have been evaluated in the context of concurrent measurement of all adrenal steroid hormones. Serum androgens, mineralocorticoids, glucocorticoids, progesterones and androgens were assessed in 70 healthy young men using ultra high performance supercritical fluid chromatography and tandem mass spectrometry. Testosterone, 24.5 nmol/L was the most prominent androgen detected in all participants while dihydrotestosterone, 1.23 nmol/L, was only detected in 25% of the participants. The 11-oxy androgens were present in most of the participants with 11-hydroxyandrostenedione, 3.37 nmol, in 98.5%, 11-ketoandrostenedione 0.764 in 77%, 11-hydroxytestosterone, 0.567 in 96% and 11-ketotestosterone: 0.440 in 63%. A third of the participants with normal testosterone and comparable 11-ketotestosterone, had significantly lower dehydroepiandrosterone (p < 0.001). In these males 11-hydroxyandrostenedione (p < 0.001), 11-ketoandrostenedione (p < 0.01) and 11-hydroxytestosterone (p < 0.006) were decreased. Glucocorticoids were also lower: cortisol (p < 0.001), corticosterone (p < 0.001), cortisone (p < 0.006) 11-dehydrocorticosterone (p < 0.001) as well as cortisol:cortisone (p < 0.001). The presence of dehydroepiandrosterone was associated with 16-hydroxyprogesterone (p < 0.001), which was also significantly lower. Adrenal and gonadal steroid analysis showed unexpected steroid heterogeneity in normal young men. Testosterone constitutes 78% of the circulating free androgens with the 11-oxy androgens abundantly present in all participants significantly contributing 22%. In addition, a subset of men were identified with low circulating dehydroepiandrosterone who showed altered adrenal steroids with decreased glucocorticoids and decreased C11-oxy androgens. Analysis of the classical and 11-oxy androgens with the additional measurement of dehydroepiandrosterone and 16-hydroxyprogesterone may allow better diagnostic accuracy in androgen excess or deficiency.

Cu-Modified Palladium Catalysts: Boosting Formate Electrooxidation via Interfacially OHad-Driven Had Removal.

Direct formate fuel cells have gained traction due to their eco-friendly credentials and inherent safety. However, their potential is hampered by the kinetic challenges of the formate oxidation reaction (FOR) on Pd-based catalysts, chiefly due to the unfavorable adsorption of hydrogen species (Had). These species clog the active sites, hindering efficient catalysis. Here, we introduce a straightforward strategy to remedy this bottleneck by incorporating Pd with Cu to expedite the removal of Pd-Had in alkaline media. Notably, Cu plays a pivotal role in bolstering the concentration of hydroxyl adsorbates (OHad) on the surface of catalyst. These OHad species can react with Had, effectively unblocking the active sites for FOR. The as-synthesized catalyst of PdCu/C exhibits a superior FOR performance, boasting a remarkable mass activity of 3.62 A mg-1. Through CO-stripping voltammetry, we discern that the presence of Cu in Pd markedly speeds up the formation of adsorbed hydroxyl species (OHad) at diminished potentials. This, in turn, aids the oxidative removal of Pd-Had, leveraging a synergistic mechanism during FOR. Density functional theory computations further reveal intensified interactions between adsorbed oxygen species and intermediates, underscoring that the Cu-Pd interface exhibits greater oxyphilicity compared to pristine Pd. In this study, we present both experimental and theoretical corroborations, unequivocally highlighting that the integrated copper species markedly amplify the generation of OHad, ensuring efficient removal of Had. This work paves the way, shedding light on the strategic design of high-performing FOR catalysts.

Loop pathways are responsible for tuning the accumulation of C19- and C22-sterol intermediates in the mycobacterial phytosterol degradation pathway.

4-Androstene-3,17-dione (4-AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) are the most important and representative C19- and C22-steroidal materials. The optimalization of sterol production with mycobacterial phytosterol conversion has been investigated for decades. One of the major challenges is that current industrial mycobacterial strains accumulate unignorable impurities analogous to desired sterol intermediates, significantly hampering product extractions and refinements. Previously, we identified Mycobacterium neoaurum HGMS2 as an efficient 4-AD-producing strain (Wang et al. in Microb Cell Fact. 19:187, 2020). Recently, we have genetically modified the HGMS2 strain to remove its major impurities including ADD and 9OH-AD (Li et al. in Microb Cell Fact. 20:158, 2021). Unexpectedly, the modified mutants started to significantly accumulate BA compared with the HGMS2 strain. In this work, while we attempted to block BA occurrence during 4-AD accumulation in HGMS2 mutants, we identified a few loop pathways that regulated metabolic flux switching between 4-AD and BA accumulations and found that both the 4-AD and BA pathways shared a 9,10-secosteroidial route. One of the key enzymes in the loop pathways was Hsd4A1, which played an important role in determining 4-AD accumulation. The inactivation of the hsd4A1 gene significantly blocked the 4-AD metabolic pathway so that the phytosterol degradation pathway flowed to the BA metabolic pathway, suggesting that the BA metabolic pathway is a complementary pathway to the 4-AD pathway. Thus, knocking out the hsd4A1 gene essentially made the HGMS2 mutant (HGMS2Δhsd4A1) start to efficiently accumulate BA. After further knocking out the endogenous kstd and ksh genes, an HGMS2Δhsd4A1 mutant, HGMS2Δhsd4A1/Δkstd1, enhanced the phytosterol conversion rate to BA in 1.2-fold compared with the HGMS2Δhsd4A1 mutant in pilot-scale fermentation. The final BA yield increased to 38.3 g/L starting with 80 g/L of phytosterols. Furthermore, we knocked in exogenous active kstd or ksh genes to HGMS2Δhsd4A1/Δ kstd1 to construct DBA- and 9OH-BA-producing strains. The resultant DBA- and 9OH-BA-producing strains, HGMS2Δhsd4A1/kstd2 and HGMS2Δkstd1/Δhsd4A1/kshA1B1, efficiently converted phytosterols to DBA- and 9OH-BA with the rates of 42.5% and 40.3%, respectively, and their final yields reached 34.2 and 37.3 g/L, respectively, starting with 80 g/L phytosterols. Overall, our study not only provides efficient strains for the industrial production of BA, DBA and 9OH-BA but also provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds.

Competitive Adsorption: Reducing the Poisoning Effect of Adsorbed Hydroxyl on Ru Single-Atom Site with SnO2 for Efficient Hydrogen Evolution.

Ruthenium (Ru) has been theoretically considered a viable alkaline hydrogen evolution reaction electrocatalyst due to its fast water dissociation kinetics. However, its strong affinity to the adsorbed hydroxyl (OHad ) blocks the active sites, resulting in unsatisfactory performance during the practical HER process. Here, we first reported a competitive adsorption strategy for the construction of SnO2 nanoparticles doped with Ru single-atoms supported on carbon (Ru SAs-SnO2 /C) via atomic galvanic replacement. SnO2 was introduced to regulate the strong interaction between Ru and OHad by the competitive adsorption of OHad between Ru and SnO2 , which alleviated the poisoning of Ru sites. As a consequence, the Ru SAs-SnO2 /C exhibited a low overpotential at 10 mA cm-2 (10 mV) and a low Tafel slope of 25 mV dec-1 . This approach provides a new avenue to modulate the adsorption strength of active sites and intermediates, which paves the way for the development of highly active electrocatalysts.

Improving the biotransformation efficiency of soybean phytosterols in Mycolicibacterium neoaurum by the combined deletion of fbpC3 and embC in cell envelope synthesis.

Biotransformation of soybean phytosterols into 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) by mycobacteria is the core step in the synthesis of adrenocortical hormone. However, the low permeability of the dense cell envelope largely inhibits the overall conversion efficiency of phytosterols. The antigen 85 (Ag85) complex encoded by fbpA, fbpB, and fbpC was proposed as the key factor in the combined catalysis of mycoloyl for producing mycolyl-arabinogalactan (m-AG) and trehalose dimycolate (TDM) in mycobacterial cell envelope. Herein, we confirmed that fbpC3 was essential for the biotransformation of trehalose monomycolate (TMM) to TDM in Mycolicibacterium neoaurum. The deficiency of this gene raised the cell permeability, thereby enhancing the steroid uptake and utilization. The 9-OHAD yield in the fbpC3-deficient 9-OHAD-producing strain was increased by 21.3%. Moreover, the combined deletion of fbpC3 and embC further increased the 9-OHAD yield compared to the single deletion of fbpC3. Finally, after 96 h of bioconversion in industrial resting cells, the 9-OHAD yield of 11.2 g/L was achieved from 20 g/L phytosterols and the productivity reached 0.116 g/L/h. In summary, this study suggested the critical role of the fbpC3 gene in the synthesis of TDM in M. neoaurum and verified the feasibility of improving the bioconversion efficiency of phytosterols through the cell envelope engineering strategy.

Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria.

4-Androstene-3,17-dione (4-AD), 1,4-androstadiene-3,17-dione (ADD) and 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD), which are important starting compounds for the synthesis of steroidal medicines, can be biosynthetically transformed from phytosterols by Mycobacterium strains. Genomic and metabolic analyses have revealed that currently available 4-AD-producing strains maintain the ability to convert 4-AD to ADD and 9OH-AD via 3-ketosteroid-1,2-dehydrogenase (KstD) and 3-ketosteroid-9α-hydroxylase (Ksh), not only lowering the production yield of 4-AD but also hampering its purification refinement. Additionally, these 4-AD industrial strains are excellent model strains to construct ADD- and 9OH-AD-producing strains. We recently found that Mycobacterium neoaurum HGMS2, a 4-AD-producing strain, harbored fewer kstd and ksh genes through whole-genomic and enzymatic analyses, compared with other strains (Wang et al. in Microbial Cell Fact 19:187, 2020). In this study, we attempted to construct an efficient 4-AD-producing strain by knocking out the kstd and ksh genes from the M. neoaurum HGMS2 strain. Next, we used kstd- and ksh-default HGMS2 mutants as templates to construct ADD- and 9OH-AD-producing strains by knocking in active kstd and ksh genes, respectively. We found that after knocking out its endogenous kstd and ksh genes, one of these knockout mutants, HGMS2Δkstd211 + ΔkshB122, showed a 20% increase in the rate of phytosterol to 4-AD conversion, compared relative to the wild-type strain and an increase in 4-AD yield to 38.3 g/L in pilot-scale fermentation. Furthermore, we obtained the ADD- and 9OH-AD-producing strains, HGMS2kstd2 + Δkstd211+ΔkshB122 and HGMS2kshA51 + Δkstd211+ΔkshA226, by knocking in heterogenous active kstd and ksh genes to selected HGMS2 mutants, respectively. During pilot-scale fermentation, the conversion rates of the ADD- and 9OH-AD-producing mutants transforming phytosterol were 42.5 and 40.3%, respectively, and their yields reached 34.2 and 37.3 g/L, respectively. Overall, our study provides efficient strains for the production of 4-AD, ADD and 9OH-AD for the pharmaceutical industry and provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds.

Whole-genome and enzymatic analyses of an androstenedione-producing Mycobacterium strain with residual phytosterol-degrading pathways.

Mycobacterium neoaurum strains can transform phytosterols to 4-androstene-3,17-dione (4-AD), a key intermediate for the synthesis of advanced steroidal medicines. In this work, we presented the complete genome sequence of the M. neoaurum strain HGMS2, which transforms ß-sitosterol to 4-AD. Through genome annotation, a phytosterol-degrading pathway in HGMS2 was predicted and further shown to form a 9,10-secosteroid intermediate by five groups of enzymes. These five groups of enzymes included three cholesterol oxidases (ChoM; group 1: ChoM1, ChoM2 and Hsd), two monooxygenases (Mon; group 2: Mon164 and Mon197), a set of enzymes for side-chain degradation (group 3), one 3-ketosteroid-1,2-dehydrogenase (KstD; group 4: KstD211) and three 3-ketosteroid-9a-hydroxylases (Ksh; group 5: KshA226, KshA395 and KshB122). A gene cluster encoding Mon164, KstD211, KshA226, KshB122 and fatty acid ß-oxidoreductases constituted one integrated metabolic pathway, while genes encoding other key enzymes were sporadically distributed. All key enzymes except those from group 3 were prepared as recombinant proteins and their activities were evaluated, and the proteins exhibited distinct activities compared with enzymes identified from other bacterial species. Importantly, we found that the KstD211 and KshA395 enzymes in the HGMS2 strain retained weak activities and caused the occurrence of two major impurities, i.e., 1,4-androstene-3,17-dione (ADD) and 9-hydroxyl-4-androstene-3,17-dione (9OH-AD) during ß-sitosterol fermentation. The concurrence of these two 4-AD analogs not only lowered 4-AD production yield but also hampered 4-AD purification. HGMS2 has the least number of genes encoding KstD and Ksh enzymes compared with current industrial strains. Therefore, HGMS2 could be a potent strain by which the 4-AD production yield could be enhanced by disabling the KstD211 and KshA395 enzymes. Our work also provides new insight into the engineering of the HGMS2 strain to produce ADD and 9OH-AD for industrial application.

Improving the biotransformation of phytosterols to 9α-hydroxy-4-androstene-3,17-dione by deleting embC associated with the assembly of cell envelope in Mycobacterium neoaurum.

The conversion of low value-added phytosterols into 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) by mycobacteria is an important step in the steroid pharmaceutical industry. However, the highly dense cell envelope with extremely low permeability largely affects the overall transformation efficiency. Here, we preliminarily located the key gene embC required for the synthesis of lipoarabinomannan from lipomannan in Mycobacterium neoaurum. The genetic manipulation of embC indicated that it might be the only functional enzyme catalyzing the above synthesis process. The deficiency of lipoarabinomannan led to a significantly increased cell permeability, which in turn caused the enhanced uptake capacity of cells. The sterol substrate conversion efficiency of mycobacterial cells was increased by about 52.4 % after 72-h conversion. Ultimately, the absence of embC increased the productivity from 0.0927 g/L/h to 0.1031 g/L/h, as confirmed by a resting cell system. This study verified the feasibility of improving the efficiency of the microbial conversion system through the cell envelope engineering strategy.

The 11ß-hydroxysteroid dehydrogenase isoforms: pivotal catalytic activities yield potent C11-oxy C19 steroids with 11ßHSD2 favouring 11-ketotestosterone, 11-ketoandrostenedione and 11-ketoprogesterone biosynthesis.

The 11ß-hydroxysteroid dehydrogenase (11ßHSD) types 1 and 2 are primarily associated with glucocorticoid inactivation and reactivation. Several adrenal C11-oxy C19 and C11-oxy C21 steroids, which have been identified in prostate cancer, 21-hydroxylase deficiency and polycystic ovary syndrome, are substrates for these isozymes. This study describes the kinetic parameters of 11ßHSD1 and 11ßHSD2 towards the C11-keto and C11-hydroxy derivatives of the C19 and C21 steroids. The apparent Km and Vmax values indicate the more prominent 11ßHSD2 activity towards 11ß-hydroxy androstenedione, 11ß-hydroxytestosterone and 11ß-hydroxyprogesterone in contrast to the 11ßHSD1 reduction of the C11-keto steroids, as was demonstrated in the LNCaP cell model in the production of 11-ketotestosterone and 11-ketodihydrotestosterone. Data highlighted the role of 11ßHSD2 and cytochrome P450 17A1 in the contribution of C11-oxy C21 steroids to the C11-oxy C19 steroid pool in the C11-oxy backdoor pathway. In addition, 11ßHSD2 activity, catalysing 11-ketotestosterone biosynthesis, was shown to be key in the production of prostate specific antigen and in the progression of prostate cancer to castration resistant prostate cancer. The study at hand thus provides evidence that 11ßHSD isozymes play key roles in pathophysiological states, more so than was previously put forward.

Enhancing Expression of 3-Ketosteroid-9α-Hydroxylase Oxygenase, an Enzyme with Broad Substrate Range and High Hydroxylation Ability, in Mycobacterium sp. LY-1.

3-Ketosteroid-9α-hydroxylase (KSH) consists of two protein systems, KshA and KshB, and is a key enzyme in microbial degradation pathway of natural sterols. 9α-Hydroxy-4-androstene-3,17-dione (9α-OH-AD) is a valuable steroid pharmaceutical intermediate. The expression of a 3-ketosteroid-9α-hydroxylase oxygenase (KshA1) with a broad substrate range and high hydroxylation ability was enhanced in Mycobacterium sp. LY-1 to improve the yield of 9α-OH-AD. Through whole-genome sequence mining and homologous comparison, the putative genes (kshA1 and kshB) in wild strain LY-1 were firstly identified. Then they were heterogeneously co-expressed in Escherichia coli BL21. The transformation results of recombinant BL21-KshA1/B demonstrated KshA1/B had high hydroxylation ability to AD. Moreover, substrate preference analysis suggested that KshA1LY-1 had a broad substrate range. After enhancing expression of kshA1 and kshB in the strain LY-1, the maximum productivity of 9α-OH-AD in recombinant LY-1-KshA1/B reached 0.064 g/L/h in a 5-L stirred fermenter.

Engineered 3-Ketosteroid 9α-Hydroxylases in Mycobacterium neoaurum: an Efficient Platform for Production of Steroid Drugs.

3-Ketosteroid 9α-hydroxylase (Ksh) consists of a terminal oxygenase (KshA) and a ferredoxin reductase and is indispensable in the cleavage of steroid nucleus in microorganisms. The activities of Kshs are crucial factors in determining the yield and distribution of products in the biotechnological transformation of sterols in industrial applications. In this study, two KshA homologues, KshA1N and KshA2N, were characterized and further engineered in a sterol-digesting strain, Mycobacterium neoaurum ATCC 25795, to construct androstenone-producing strains. kshA1N is a member of the gene cluster encoding sterol catabolism enzymes, and its transcription exhibited a 4.7-fold increase under cholesterol induction. Furthermore, null mutation of kshA1N led to the stable accumulation of androst-4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD). We determined kshA2N to be a redundant form of kshA1N Through a combined modification of kshA1N, kshA2N, and other key genes involved in the metabolism of sterols, we constructed a high-yield ADD-producing strain that could produce 9.36 g liter-1 ADD from the transformation of 20 g liter-1 phytosterols in 168 h. Moreover, we improved a previously established 9α-hydroxy-AD-producing strain via the overexpression of a mutant KshA1N that had enhanced Ksh activity. Genetic engineering allowed the new strain to produce 11.7 g liter-1 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) from the transformation of 20.0 g liter-1 phytosterol in 120 h.IMPORTANCE Steroidal drugs are widely used for anti-inflammation, anti-tumor action, endocrine regulation, and fertility management, among other uses. The two main starting materials for the industrial synthesis of steroid drugs are phytosterol and diosgenin. The phytosterol processing is carried out by microbial transformation, which is thought to be superior to the diosgenin processing by chemical conversions, given its simple and environmentally friendly process. However, diosgenin has long been used as the primary starting material instead of phytosterol. This is in response to challenges in developing efficient microbial strains for industrial phytosterol transformation, which stem from complex metabolic processes that feature many currently unclear details. In this study, we identified two oxygenase homologues of 3-ketosteroid-9α-hydroxylase, KshA1N and KshA2N, in M. neoaurum and demonstrated their crucial role in determining the yield and variety of products from phytosterol transformation. This work has practical value in developing industrial strains for phytosterol biotransformation.

Enhancement of 9α-Hydroxy-4-androstene-3,17-dione Production from Soybean Phytosterols by Deficiency of a Regulated Intramembrane Proteolysis Metalloprotease in Mycobacterium neoaurum.

Modification of the sterol catabolism pathway in mycobacteria may result in the accumulation of some valuable steroid pharmaceutical intermediates, such as 9α-hydroxy-4-androstene-3,17-dione (9-OHAD). In previous work, sigma factor D (SigD) was identified as a negative factor of the 9-OHAD production in Mycobacterium neoaurum. Here, the deficiency of rip1 putatively coding for a regulated intramembrane proteolysis metalloprotease (Rip1), which could cleave the negative regulator of SigD (anti-SigD), enhanced the transcription of some key genes (choM1, kshA, and hsd4A) in the sterol catabolic pathway. Furthermore, the deletion of rip1 increased the consumption of phytosterols by 37.8% after 96 h of growth in M. neoaurum. The production of 9-OHAD in the engineered M. neoaurumΔkstD1ΔkstD2ΔkstD3Δrip1 (MnΔk123Δrip1) strain was ultimately increased by 27.3% compared to that in its parental strain M. neoaurumΔkstD1ΔkstD2ΔkstD3 (MnΔk123). This study further confirms the important role of SigD-related factors in the catabolism of sterols.

[Mutation breeding of high 9α-hydroxy-androst-4-ene-3,17- dione transforming strains from phytosterols and their conversion process optimization].

In order to improve transformation efficiency of phytosterols into 9α-hydroxylation of 4-androstene-3,17-dione (9α-OH-AD) by Mycobacterium sp. LY-1, we studied the strains breeding using atmospheric and room temperature plasma (ARTP) technology and optimized their conversion process. A high production strain named C33 with a good genetic stability was selected and the product molar yield reached to 15.5%, 34.8% higher than that of original strain with 15 g/L phytosterols. Furthermore, the fermentation medium was optimized through the design of orthogonal experiment. Besides, oil-water bidirectional transformation system was set up to improve the 9α-OH-AD molar yield of mutant strain C33. With adding 12 mL soybean oil to each 1 g phytosterols, the molar yield of 9α-OH-AD reached 47.0%, which increased twice than that of control (15.5%).

Analysis of Intermediates of Steroid Transformations in Resting Cells by Thin-Layer Chromatography (TLC).

Thin-layer chromatography (TLC) is a useful and convenient method for the analysis of steroids due to: its simple sample preparation, low time-consuming process, high sensitivity, low equipment investment and capacity to work on many samples simultaneously. Here we describe a TLC easy protocol very useful to analyze steroid molecules derived from a biotransformation carried out in wild-type and mutant resting cells of Rhodococcus ruber strain Chol-4. Following this protocol, we were able to detect the presence or the absence of some well-known intermediates of cholesterol catabolism in Rhodococcus, namely AD, ADD, and 9OHAD.

Role Identification and Application of SigD in the Transformation of Soybean Phytosterol to 9α-Hydroxy-4-androstene-3,17-dione in Mycobacterium neoaurum.

9α-Hydroxy-4-androstene-3,17-dione (9-OHAD) is a valuable steroid pharmaceutical intermediate which can be produced by the conversion of soybean phytosterols in mycobacteria. However, the unsatisfactory productivity and conversion efficiency of engineered mycobacterial strains hinder their industrial applications. Here, a sigma factor D (sigD) was investigated due to its dramatic downregulation during the conversion of phytosterols to 9-OHAD. It was determined as a negative regulator in the metabolism of phytosterols, and the deletion of sigD in a 9-OHAD-producing strain significantly enhanced the titer of 9-OHAD by 18.9%. Furthermore, a high yielding strain was constructed by the combined modifications of sigD and choM2, a key gene in the phytosterol metabolism pathway. After the modifications, the productivity of 9-OHAD reached 0.071 g/L/h (10.27 g/L from 20 g/L phytosterol), which was 22.5% higher than the original productivity of 0.058 g/L/h (8.37 g/L from 20 g/L phytosterol) in the industrial resting cell biotransformation system.

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.