Cascade biotransformation of estrogens by Isaria fumosorosea KCh J2

Estrone, estradiol, ethynylestradiol and estrone 3-methyl ether underwent a biotransformation process in the submerged culture of Isaria fumosorosea KCh J2. Estrone was transformed into seven metabolites, four of which were glycosylated. Estradiol was selectively glycosylated at C-3 and then transformed to D-ring lactone. Ethynylestradiol was coupled with methylglucoside and 6β-hydroxyderivative was obtained. Estrone 3-methyl ether was not transformed indicating that a free hydroxyl group at C-3 is necessary for glycosylation. Baeyer–Villiger oxidation combined with hydroxylation and glycosylation was observed. All glycosides obtained in this study are 3-O-β-methylglucosides.

Preparative biotransformation. The same transformations were performed on the preparative scale in 2000 mL flasks, each containing 500 mL of the cultivation medium. The culture of I. fumosorosea KCh J2 was incubated under the same conditions as in the screening procedure, and then 100 mg of substrate dissolved in 2 mL of DMSO was added to the 3-day-old culture. After the complete transformation of the substrate, the mixture was extracted with ethyl acetate (3 × 300 mL), dried (anhydrous MgSO 4 ) and concentrated in vacuo. The crude mixture obtained this way was separated by preparative TLC and analysed (TLC, HPLC).

Results and Discussion
Spectral data of isolated metabolites. 6β-hydroxyestrone (5). 1  3,6β-dihydroxy-17a-oxa-D-homo-estrone (7). 1 (8). 1 (10). 1 (11). 1  www.nature.com/scientificreports www.nature.com/scientificreports/   (14). 1 (15). 1 (16). 1  Transformation of estrone (1) in the culture of Isaria fumosorosea KCh J2 led to seven metabolites. Four of them were obtained as methyloglucosyl derivatives (Fig. 1). Substrate 1 was hydroxylated at the 6β position to compound 5. Then the C-17 carbonyl group of 5 was reduced, giving compound 6. Estradiol was not observed in the reaction mixture, which disproves the possibility of reducing estrone to estradiol and then hydroxylating it at the 6β position. It can be assumed that the dehydrogenases reducing the carbonyl group at C-17 accepted only 6β-hydroxy derivative. Simultaneously, the C-17 carbonyl group of 5 is necessary for Baeyer-Villiger oxidation of the D ring, which led to 7. Apparently the hydroxyl group at the 6β position has to be a steric hindrance for glycosylation because none of the 6β-hydroxylated derivatives was conjugated with a glycosyl moiety. The substrate (1) was glycosylated at the C-3 hydroxyl group, giving compound 8, which was transformed further to D-ring lactone 9. The tested strain can introduce a double bond between C-9 and C-11 in the obtained lactone, forming C-9 unsaturated D-lactone 10. Together with that transformation, hydroxylation of 8 at the C-2 position occurred, giving product 11. No free 2-hydroxyestrone and no conjugated 6β-hydroxy-derivatives were detected, which suggests that the methylglucosyl moiety is a steric hindrance for 6β-steroid hydroxylase but not for 2-steroid hydroxylase. Furthermore, Baeyer-Villiger oxidation combined with the previous hydroxylation is not common for microbial steroid transformation. Such a combination of reactions is possible in Beauveria bassiana 22 www.nature.com/scientificreports www.nature.com/scientificreports/ Transformation of estradiol (2), estrane with two free hydroxyl groups at the C-3 and C-17β position, was performed to evaluate regioselectivity of glycosylation. As in the case of estrone (1), free 6β-hydroxy derivatives were obtained. Lactone 7 was a result of 6β-hydroxylation of 2, then oxidation of the C-17β hydroxyl group to 5 and then Baeyer-Villiger oxidation (Fig. 2). Simultaneously, regioselective glycosylation to 12 occurred at the C-3 position of 2. Then, the C-17β hydroxyl group of conjugated 12 was oxidised twice -to compound 8 and then to 9. At the same time, between C-9 and C-11 of 12 a double bond was introduced, forming 13, then compound 13 was oxidized to give 14. Probably, compound 14 can be formed from 8 too. Similar to estrone (1), Isaria fumosorosea KCh J2 is not able to glycosylate 6β-hydroxyderivatives of estradiol (2). This substrate is metabolized faster than estrone (1), but emerging products are in nearly equal concentrations.

3-(β-D-4′-O-methyloglucosyloxy)-17-ethynyloestr-17β-ol
The multitude of emerging products of estrone (1) and estradiol (2) is the result of glycosylation and transformations in the D-ring of those compounds. Therefore ethynylestradiol (3), with a relatively unreactive substituent at C-17, was used. As expected, 3 was transformed into two products (Fig. 3). One was a free 6β-hydroxy derivative 15, and the other one was a methylglucosyl derivative 16. The ethynyl group at the 17α position makes oxidation of the 17α-hydroxyl group impossible, which explains the lack of Baeyer-Villiger oxidation products.  www.nature.com/scientificreports www.nature.com/scientificreports/ 3-Methoxyestrone (4) was used to assess whether O-demethylation to estrone (1), as in flavone compounds 25 or only D-ring transformation, similar to estrone (1), occurs. Surprisingly, 4 was not transformed in the I. fumosorosea KCh J2 culture. Inhibition of any activity toward this substrate was observed. A free hydroxyl group at C-3 is necessary for transformation of 3-methoxyestrone (4) by this strain.
The transformation course for all presented substrates was tested using the HPLC technique. Because of the multitude of products from substrates 1-3 and their poor separation, it was impossible to determine the percentage composition of the mixture unambiguously. Additionally, NMR with the internal standard was used to establish the transformation pathway for substrates 1-3. In this case, the whole transformation broth was extracted, evaporated and dissolved in a deuterated solvent, but the number of products and their similar spectral data caused the experiment challenging to analyse. However, the amount of unreacted substrate and approximate composition of the products was estimated for all cases (Fig. 4). Taking into account the results of the two methods of tracking the transformation course, it can be said that the transformation of estrone (1) in I. fumosorosea KCh J2 took three days and the main product obtained was 8 which composition in the crude mixture was over 60%. However, after a longer time, a gradual decrease in the amount of this compound is observed due to its conversion into subsequent glycosidic products. Among them, 3-(β-D-4′-O-methyloglucosyloxy)-17a-oxa-D-homo-estr-17-one (9) was 16% of the crude mixture after 3 days of transformation. The maximum concentration of 9 was observed after ten days, and it reached 25%. The rest of the glycosidic products were in concentration between 1 and 8%. The percentage of products without a glycosidic group did not exceed 10%.
Transformation of estradiol (2) was faster, whole added substrate being converted in less than 24 hours, but the obtained products were in nearly equal concentrations. None of the products was in the majority, like in estrone (1) transformation. Noteworthy is also definitely a higher percentage of products without a glycosidic group. During the biotransformation estradiol (2) their share was recorded at 30%.
In the case of (3), after 24 hours of biotransformation, 6% of the substrate was observed by NMR technique, and the whole conversion occurred in 7 days. During the incubation of this substrate in the culture of the test strain, a constant ratio of both products (determined as 85 to 15% for compounds 16 and 15, respectively) was observed.
The metabolic pathways of the tested compounds described above have been proposed both based on isolated products and the transformation course analysed by HPLC or NMR. The preparation process of glycoside analogues and their separation needs further development.
Steroid glycosides are known as biotransformation products obtained from the cultures of Syncephalastrum racemosum AS 3.264 26 , Mucor hiemalis 27 and many plants 28 . Glucosyloestrogens were obtained in the cultures of Rhizopus oryzae AS 3.2380 14 and microalgae of the genus Selenastrum 15 . In the culture of R. oryzae AS 3.2380 estrogen 3β-glucosides were separated but no further transformation was observed. 96-hour transformation of Selenastrum capricornutum produced glucosides of 2-hydroxy and 6β-hydroxyethynylestradiol, but they were in   www.nature.com/scientificreports www.nature.com/scientificreports/ the minority (both 5%). 6β-Hydroxyethynylestradiol was also obtained in the transformation in the culture of Cephalosporium aphidicola and Cunninghamella elegans 29 and the alga Ankistrodesmus braunii 15 , but no glucosides were detected. To the best of the authors' knowledge, this is the first description of fungal catalysed methylglycosylation of estrone derivatives and their further transformation.
The reaction of methylglycosylation is usually described for strains of the species Beauveria bassiana. The ability of strains from this species to catalyze this reaction for a wide range of substrates (in addition to steroids) has been investigated [30][31][32][33][34] .
It has recently been demonstrated that also other entomopathogenic strains such as Isaria fumosorosea and I. farinosa are capable of attaching a 4-O-methylglucose moiety, however, until now, such a transformation has been described only for flavonoid substrates 16,17,25 .

Conclusions
Biotransformations of steroids using Isaria fumosorosea KCh J2 are a valuable source of many derivatives. Estrone derivatives obtained in this study are products of the multienzyme activity of this strain: hydroxylase, reductase, oxidase and glucosyltransferase. All glycosyls obtained in this study are 3-O-β-D-(4′-O-methyl)-glucopyranos ides. Transformations of estradiol, estrone, ethinyloestradiol and methoxyestradiol were performed to evaluate the regioselectivity of glycosylation. Interesting features have been elucidated through this work. First of all, the hydroxyl group at the 6β position most likely is a steric hindrance for glucosylation, because none of the 6β-hydroxylated derivatives was conjugated with a glycosyl moiety. Second, no free 2-hydroxyestrone, as well as conjugated 6β-hydroxy-derivatives, were detected, which suggests that the methylglucosyl moiety is a steric hindrance for 6β-steroid hydroxylase but not for 2-steroid hydroxylase. Third, glycosylated derivatives are more likely to be transformed further, including to lactones. To the best of our knowledge, this is the first demonstration of further transformation of glycosylated estrogens by whole fungal cells.