The introduction of the 9α-hydroxy group into androst-4-en-3,17-dione using a new actinobacterium strain

Rodina, N. V.; Andryushina, V. A.; Stytsenko, T. S.; Turova, T P; Baslerov, R. V.; Panteleeva, A. N.; Voishvillo, N. E.

doi:10.1134/s0003683809040085

Cited by 9 publications

(6 citation statements)

References 17 publications

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“…A possible reason for the lack of any undesirable 9α-OH-AD degradation (which was observed at the AD load of 5 g/l) during the AD transformation at a load of 10 g/l is that the increased AD load resulted in the accumulation of 9α-OH-AD in the culture broth in the form of crystal-like structures (Fig. 3) unavailable for the further microbial destruction (Rodina et al, 2009). During this study we selected optimal conditions providing AD transformation at a load of 30 g/L and obtaining a high output of a technical product.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, actinobacteria capable of providing a high-selective introduction of a hydroxy group at the 9α-position of a steroid molecule may be considered as the most promising bioreagents, since the corresponding bacterial enzyme shows the lower substrate specificity than steroid 9α-monooxygenase of a fungal origin (Petrusma et al, 2009(Petrusma et al, , 2011Lee et al, 2016;Nielsen & Keasling, 2016). However, 9α-hydroxylation activity was detected only in bacteria which were able to use steroids as a carbon source since this reaction represents an intermediate stage of a complete cleavage of steroid molecules to CO 2 and H 2 O, at which the simultaneous action of 3-ketosteroid-1,2-dehydrogenase and 9α-hydroxylase is observed (Petrusma et al, 2014;Donova & Egorova, 2012;Rodina et al, 2009). The analysis of published data showed that the selective 9α-hydroxylation without any destruction of a steroid nucleus can be performed using bacterial strains carrying mutations, which block the biosynthesis of 1,2-dehydrogenase or prevent the functioning of this enzyme.…”

Section: Introductionmentioning

confidence: 99%

“…The analysis of published data showed that the selective 9α-hydroxylation without any destruction of a steroid nucleus can be performed using bacterial strains carrying mutations, which block the biosynthesis of 1,2-dehydrogenase or prevent the functioning of this enzyme. In our previous study, we obtained a highly-selective Rhodococcus erythropolis strain VKPM AC-1740 with improved 9α-hydroxylating activity (Rodina et al, 2009;Carpova-Rodina et al, 2011). Using this strain, we developed efficient technologies for the production of 9α-hydroxy-steroid derivatives (Vojshvillo et al, 2007) and developed several efficient methods for 9α-OH-AD production from sterols including the mixed culture method (Andryushina et al, 2011) or the use of original biocatalyst representing R. erythropolis VKPM AC-1740 cells immobilized in a PVA cryogel (Carpova- Rodina et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

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

Andryushina¹,

Karpova²,

Stytsenko³

et al. 2018

Regul. Mech. Biosyst.

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Andryushina¹,

Karpova²,

Stytsenko³

et al. 2018

Regul. Mech. Biosyst.

Self Cite

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“…The development of environmentally friendly downstream procedures for end products formed during the biotransformation in the presence of CD is of great importance. As a rule, steroids extracted with aprotic water‐immiscible organic solvents, where the transition of CD derivatives into organic phase is excluded 16‐20 . Another approach is based on the adsorption of steroid products by polymer carriers followed by treatment with organic solvents 21,22 …”

Section: Introductionmentioning

confidence: 99%

Novel approach for downstream processing of phytosterol bioconversion

Хомутов

Шутов

Averin

et al. 2023

J of Chemical Tech & Biotech

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BACKGROUNDDownstream processing is an important part of industrial steroid biotechnology. The traditional recovery methods require significant amounts of the organic solvents which are associated with environmental risks. There is a need for environmentally friendly downstream processing which also may open the prospects for regeneration and repeated use of the costly materials.RESULTSMicrobial conversion of phytosterol to C17‐ketosteroids in methylated cyclodextrin (mCD) solutions was studied as a model bioprocess. Quantitative aspects of competitive complexation of mCD and natural β‐CD (β‐CD) with steroids were studied under different temperature modes and CD concentrations. The method of steroid recovery based on precipitation of insoluble β‐CD complexes ensured the complete isolation of androst‐4‐ene‐3,17‐dione, androsta‐1,4‐diene‐3,17‐dione, 9α‐hydroxyandrost‐4‐ene‐3,17‐dione, 20‐hydroxymethyl‐pregna‐1,4‐dien‐3‐one and 3β‐hydroxyandrost‐5‐en‐17‐one from aqueous media. Analysis of the 1H nuclear magnetic resonance (NMR) spectra showed a constant molar ratio of β‐CD to steroid equal to 2 in the obtained steroid β‐CD complexes. The extraction of the steroid from the β‐CD complex by small volumes of organic solvent provided a quantitative yield. The method developed ensures regeneration and repeated use of the mCDs without decrease of the basic bioconversion outputs, as was demonstrated in a series of phytosterol to androsta‐1,4‐diene‐3,17‐dione bioconversions by Mycolicibacterium neoaurum VKM Ac‐1816D.CONCLUSIONSDownstream processing based on manipulation of steroid solubility in aqueous cyclodextrin solutions is a promising alternative to traditional steroid extraction with organic solvents. This environmentally friendly approach ensures re‐use of materials without decrease in bioconversion outputs. © 2023 Society of Chemical Industry (SCI).

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“…However, the main objectives in the research and development of the steroid drug industry currently consist of the detection and isolation of microbial strains with novel activity or more efficient transformation capacity, where genetic engineering and metabolic engineering can play a prominent role in the metabolism of bacteria, fungi, and plants [33][34][35][36].…”

Section: Introductionmentioning

confidence: 99%

Biotransformation of Steroids Using Different Microorganisms

Cano-Flores¹,

Gómez²,

Ramos³

2020

Chemistry and Biological Activity of Steroids

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The introduction of a hydroxyl group "biohydroxylation" in the steroid skeleton is an important step in the synthesis of new steroids used physiologically as hormones and active drugs. There are currently about 300 known steroid drugs whose production constitutes the second category within the pharmaceutical market after antibiotics. Several biotransformations at industrial scale have been applied in the production of steroid hormones and drugs, which have functionalized different types of raw materials by means of chemo-, regio-, and stereoselective reactions (hydroxylation, Baeyer-Villiger oxidation, oxidation reactions, reduction of group carbonyl, isomerization, and Michael additions, condensation reactions, among others). In Green Chemistry, biotransformations are an important chemical methodology toward more sustainable industrial processes.

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The introduction of the 9α-hydroxy group into androst-4-en-3,17-dione using a new actinobacterium strain

Cited by 9 publications

References 17 publications

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

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

Novel approach for downstream processing of phytosterol bioconversion

Biotransformation of Steroids Using Different Microorganisms

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