Optimization of the 9α-hydroxylation of steroid substrates using an original culture of Rhodococcus erythropolis

Keywords: fluorinated corticoids; AD; 9α-ОН-АD; Rhodococcus erythropolis VKPM AC-1740; surfactants; inducer.


To obtain inoculation material (cultivation stage 1), the biomass of Rhodococcus erythropolis VKPM AC-1740 was transferred from agar slants into 750 ml conic flasks containing 100 ml of vegetation media of the following composition (g/l): medium 1 – yeast extract, 10.0; glucose, 10.0; soybean flour, 10.0; КН2РО4, 2.0; Na2НРО4, 4.0 (рН 6.8–7.4); medium 2 – corn extract, 15.0; glucose, 10.0; КН2РО4, 2.0; Na2НРО4, 4.0 (рН 6.8–7.4). The culture was grown on a rotary shaker (220 rpm) for 68–72 h at 28–29 °С. To obtain a working biomass (cultivation stage 2), the inoculum obtained at the stage 1 was transferred into flasks containing the same media (the volume of seed material was 20% of the medium volume) and grown under the same conditions for 23–25 h. During a study of the effect of the inducer concentration on the rate of 9α-OH-AD formation, different concentrations (0.25, 0.50, and 1 g/l) of the AD solution in dimethylformamide (DMF) were added to the vegetation medium after 6 h of incubation. To perform AD transformation at a load of 5 g/l, 10 ml of Rh. erythropolis cells at the age of 23–25 h were transferred into 750 mL flasks with baffles containing 40 mL of vegetation medium supplemented with the steroid. AD was added in the form of microcrystals or suspension with a surfactant or DMF. The process was carried out at 28–29 ºC and with constant mixing (220 rpm). During AD transformation at a load of 10–30 g/l, the steroid was preliminarily precipitated from DMF solution. The resulting paste was mixed with a surfactant and transformation medium. The obtained homogeneous suspension was poured in equal amounts into the flasks with baffles, and then a concentrated cell mass was added (25 vol.%). To obtain a cell concentrate, cells were centrifuged for 1 h at 1500 rpm at the age of 23–25 h. The resulting biomass was homogenized, supplemented with a fresh medium to the required volume, and added into transformation flasks. The amount of a biomass required for AD transformation at a load of 10 g/l was 3.13 g/l (dry weight); in the case of a 30 g/l load, the biomass was added by two equal portions, and its total amount was 6.2 g/l (dry weight). The amount of 9α-OH-AD in a culture broth was evaluated by a thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Steroids were extracted by ethylacetate. To perform TLC, Sorbifil plates (Russia) and benzol: acetone mix (3 : 1) were used. HPLC was performed on a Gilson chromatographer (United States) equipped with a Silasorb C-18 column (10 μm, 4.0 × 250 mm); the flow rate was 0.8 ml/min. The mobile phase was МеОН : Н2О mix (70 : 30). The absorbance was measured at 260 nm. Replacement of corn extract, which has an unstable composition, by yeast extract and soybean flour and the use of glucose as an optimal carbon source for a Rh. erythropolis culture have provided a high-yield production of 9α-hydroxy-4-ene-3,17-dione with increased AD loads. Use of such techniques as the inoculum induction and application of surfactants have provided a positive effect on the AD transformation with a load exceeding 10 g/l. During 9α-hydroxylation of AD with a load of 30 g/l, a target product with the yield of 83% has been obtained.


Akhrem, A. A., & Titov, Y. A. (1970). Steroids and microorganisms. Nauka, Moscow (in Russian).

Andryushina, V. V, Stytsenko, T. S., Voishvillo, N. E., Iaderetz, V. V., & Druzhinina, A. V. (2013). Effect of the steroid molecule structure on the direction of its hydroxylation by the fungus Curvularia lunata. Applied Biochemistry and Microbiology, 49(4), 382–390.

Angelova, B., Mutafov, S., Avramova, T., Dimova, I., & Boyadjie, L. (1996). 9α-Hydroxylation of 4-androstene-3,17-dione by resting Rhodococcus sp. cells. Process Biochemistry, 31(2), 179–184.

Arnell, R., Johannisson, R., Lindholm, J., Fornstedt, T., Ersson, B., Ballagi, A., & Caldwell, K. (2007). Biotechnological approach to the synthesis of 9alpha-hydroxylated steroids. Preparative Biochemistry and Biotechnology, 3, 309–321.

Avramova, T., Spassova, D., Mutafov S., Momchilova S., Boyadjieva, L., Damyanova, B., & Angelova, B. (2010). Effect of Tween 80 on 9a-steroid hydroxylating activity and ultrastructural characteristics of Rhodococcus sp. cells. World Journal of Microbiology and Biotechnology, 26, 1009–1014.

Barredo, J.-L., & Herráiz, I. (Eds.). (2017). Microbial steroids: Methods and protocols. Methods in Molecular Biology. Springer Science + Business Media LLC, 1645, 373.

Bhatti, H. N., & Khera, R. A. (2012). Biological transformations of steroidal compounds: A review. Steroids, 77, 1267–1290.

Brzezinska, M., Szulc, I., Brzostek, A., Klink, M., Kielbik, M., Sulowska, Z., Pawelczyk, J., & Dziadek, J. (2013). The role of 3-ketosteroid 1(2)-dehydrogenase in the pathogenicity of Mycobacterium tuberculosis. BMC Microbiology, 13, 43.

Carpova-Rodina, N. V., Andryushina, V. A., Yaderetz, V. V., Druzhinina, A. V., Stytsenko, T. S., Shaskol'skiy, B. L., Lozinsky, V. I., Huy Luu, D., & Voishvillo, N. E. (2011). Transformation of Δ4-3-ketosteroids by free and immobilized cells of Rhodococcus erythropolis actinobacterium. Applied Biochemistry and Microbiology, 47(4), 386–392.

Donova, M. V. (2017). Steroid bioconversions. Methods in Molecular Biology, 1645, 1–13.

Egorova, O. V., Nikolayeva, V. M., Sukhodolskaya, G. V., & Donova, M. V. (2009). Transformation of C19-steroids and testosterone production by sterol-transforming strains of Mycobacterium sp. Journal of Molecular Catalazysis B: Enzymatic, 57, 198–203.

Fernández de las Heras, L., van der Geize, R., Drzyzga, O., Perera, J., & Navarro-Llorens, M. J. (2012). Molecular characterization of three 3-ketosteroid-Δ(1)-dehydrogenase isoenzymes of Rhodococcus ruber strain Chol-4. Journal of Steroid Biochemistry and Molecular Biology, 132, 271–281.

Fernandez-Cabezon, L., Galán, B., & García, J. L. (2018). New insights on steroid biotechnology. Frontiers in Microbiology, 9, 958.

Guevara, G., Fernández de las Heras, L., Perera, J., Navarro, M., & Llorens, J. (2017b). Functional differentiation of 3-ketosteroid Δ1-dehydrogenase isozymes in Rhodococcus ruber strain Chol-4. Microbial Cell Factories, 16(1), 42.

Guevara, G., Heras, L. F. L., Perera, J., & Llorens, J. M. N. (2017a). Functional differentiation of 3-ketosteroid 9α-hydroxylases in Rhodococcus ruber strain Chol-4. Journal of Steroid Biochemistry and Molecular Biology, 7, 176–187.

Haußmann, U., Wolters, D. A., Fränzel, B., Eltis, L. D., & Poetsch, A. (2013). Physiological adaptation of the Rhodococcus jostii RHA1 membrane proteome to steroids as growth substrates. Journal of Proteome Research, 12, 1188–1198.

Jakočiunas, T., Jensen, M. K., & Keasling, J. D. (2016). CRISPR/Cas9 advances engineering of microbial cell factories. Metabolic Engineering, 34, 44–59.

Knol, J., Bodewits, K., Hessels, G. I., Dijkhuizen, L., & van der Geize, R. (2008). 3-Keto-5alpha-steroid delta(1)-dehydrogenase from Rhodococcus erythropolis SQ1 and its orthologue in Mycobacterium tuberculosis H37Rv are highly specific enzymes that function in cholesterol catabolism. Biochemistry Journal, 410, 339–346.

Lee, J. Y., Na, Y. A., Kim, E., Lee, H. S., & Kim, P. (2016). The actinobacterium Corynebacterium glutamicum, an industrial workhorse. Journal of Microbiology and Biotechnology, 26, 807–822.

Li, H., Sun, J., & Xu, Z. (2017). Biotransformation of DHEA into 7α,15α-diOH-DHEA. Methods in Molecular Biology, 1645, 289–295.

Liu, Y., Shen, Y., Qiao, Y., Su, L., Li, C., & Wang, M. (2016). The effect of 3-ketosteroid-Δ(1)-dehydrogenase isoenzymes on the transformation of AD to 9α-OH-AD by Rhodococcus rhodochrous DSM 43269. Journal of Industrial Microbiology and Biotechnology, 43, 1303–1311.

Marques, M. P. C., Carvalho, F., de Carvalho, C. C. C. R., Cabral, J. M. S., & Fernandes, P. (2010). Steroid bioconversion: Toward green processes. Food and Bioproduct Processing, 88, 12–20.

Mashkovskiy, M. D. (2012). Medicinal products. New Wave, Moscow (in Russian).

Mohn, W. W., Wilbrink, M. H., Casabon, I., Stewart, G. R., Liu, J., van der Geize, R., & Eltis, L. D. (2012). Gene cluster encoding cholate catabolism in Rhodococcus spp. Journal of Bacteriology, 194, 6712–6719.

Mondaca, M. A., Vidal, M., Chamorro, S., & Vidal, G. (2017). Selection of biodegrading phytosterol strains. Methods in Molecular Biology, 1645, 143–150.

Murphy, K. C., Papavinasasundaram, K., & Sassetti, C. M. (2015). Mycobacterial recombineering. Methods in Molecular Biology, 1285, 177–199.

Mutafova, B., Mutafov, S., Fernandes, P., & Berkov, S. (2016). Microbial transformations of plant origin compounds as a step in preparation of highly valuable pharmaceuticals. Journal of Drug Metabolism and Toxicology, 7(2), 1–11.

Nielsen, J., & Keasling, J. D. (2016). Engineering cellular metabolism. Cell, 164, 1185–1197.

Petrusma, M., Dijkhuizen, L., & van der Geize, R. (2009). Rhodococcus rhodochrous DSM 43269 3-ketosteroid 9alpha-hydroxylase, a two-component iron-sulfur-containing monooxygenase with subtle steroid substrate specificity. Applied and Environmental Microbiology, 75, 5300–5307.

Petrusma, M., Hessels, G., Dijkhuizen, L., & van der Geize, R. (2011). Multiplicity of 3-ketosteroid-9α-hydroxylase enzymes in Rhodococcus rhodochrous DSM 43269 for specific degradation of different classes of steroids. Journal of Bacteriology, 193, 3931–3940.

Petrusma, M., van der Geize, R., & Dijkhuizen, L. (2014). 3-Ketosteroid 9a-hydroxylase enzymes: Rieske non-heme monooxygenases essential for bacterial steroid degradation. Antonie van Leeuwenhoek, 106, 157–172.

Ribeiro, A. L., Sánchez, M., Hidalgo, A., & Berenguer, J. (2017). Stabilization of enzymes by using thermophiles. Methods in Molecular Biology, 1645, 297–312.

Rodina, N. V., Andryushina, V. V, Stytsenko, T. S., Turova, T. P., Baslerov, R. V., Panteleeva, A. N., & Voishvillo, N. E. (2009). The introduction of the 9α-hydroxy group into androst-4-en-3,17-dione using a new actinobacterium strain. Applied Biochemistry and Microbiology, 45(4), 395–400.

Smitha, M. S., Singh, S., & Singh, R. (2017). Microbial biotransformation: A process for chemical alterations. Journal of Bacteriology and Mycology, 4(2), 85.

Tsitko, I. V., Zaitsev, G. M., & Lobanok, A. G. (1999). Effect of aromatic compounds on cellular fatty acid composition of Rhodococcus opacus. Applied and Environmental Microbiology, 65(2), 853–855.

van der Geize, R., Hessels, G. I., Nienhuis-Kuiper, M., & Dijkhuizen, L. (2008). Characterization of a second Rhodococcus erythropolis SQ1 3-ketosteroid 9alpha-hydroxylase activity comprising a terminal oxygenase homologue, KshA2, active with oxygenase-reductase component KshB. Applied and Environmental Microbiology, 74, 7197–7203.

van der Geize, R., Hessels, G. I., van Gerwen, R., van der Meijden, P., & Dijkhuizen, L. (2001). Unmarked gene deletion mutagenesis of kstD, encoding 3-ketosteroid delta1-dehydrogenase, in Rhodococcus erythropolis SQ1 using sacB as counter-selectable marker. FEMS Microbiological Letters, 205, 197–202.

van der Geize, R., Hessels, G. I., van Gerwen, R., Vrijbloed, J. W., van Der Meijden, P., & Dijkhuizen, L. (2000). Targeted disruption of the kstD gene encoding a 3-kestosteroid Δ(1)-dehydrogenase isoenzyme of Rhodococcus erythropolis strain SQ1. Applied and Environmental Microbiology, 66, 2029–2036.

Voishvillo, N. E., Rodina, N. V., Andrjushina, V. A., Stytsenko, T. S., & Skryabin, K. G. (2007). Strain Rhodococcus erythropolis VKPM Ac-1740 for 9alpha-hydroxysteroids. Patent RU 2351645 (in Russian).

Wei, J. H., Yin, X., & Welander, P. V. (2016). Sterol synthesis in diverse bacteria. Frontiers in Microbiology, 7, 990.

Wilbrink, M. H., Petrusma, M., Dijkhuizen, L., & van der Geize, R. (2011). FadD19 of Rhodococcus rhodochrous DSM 43269, a steroid-coenzyme. A ligase essential for degradation of C-24 branched sterol side chains. Applied and Environmental Microbiology, 77, 4455–4464.

Yeh, C. H., Kuo, Y. S., Chang, C. M., Liu, W. H., Sheu, M. L., & Meng, M. (2014). Deletion of the gene encoding the reductase component of 3-ketosteroid 9α-hydroxylase in Rhodococcus equi USA-18 disrupts sterol catabolism, leading to the accumulation of 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid and 1,4-androstadiene-3,17-dione. Microbial Cell Factories, 13, 130.

How to Cite
Andryushina, V. A., Karpova, N. V., Stytsenko, T. S., Yaderets, V. V., Voskresenskaya, E. D., & Dzhavakhia, V. V. (2018). Optimization of the 9α-hydroxylation of steroid substrates using an original culture of Rhodococcus erythropolis. Regulatory Mechanisms in Biosystems, 9(3), 430-434. https://doi.org/https://doi.org/10.15421/021864

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